Method of production of graft co-polymer excipient with a superior peptide and protein binding property

ABSTRACT

Provided herein is a process of preparing a semi-random graft co-polymer, the product of which is difficult to fully characterize chemically. The product of the present disclosure has unique and useful properties of 1) binding to a peptide and 2) upon co-administration of the product and the peptide into animals the product prolongs the blood circulation time and elevates the level of the peptide, compared to the peptide alone without the product of the disclosure.

BACKGROUND

The development of new drug formulations for physiologically activepeptides and proteins is focused on maintaining biological activity buteven these are limited by the inherently short half-life or instabilityof the peptides and proteins in the body. This is especially true forsmall peptides and proteins with a hydrodynamic diameter of less thanabout 5 nm. There has long been a desire to alleviate such shorthalf-life or instability of peptides and proteins in the body either bythe use of infusion devices that constantly deliver rapidly degradingpeptide or protein drugs or by providing an eroding depot of the drugunder the skin. Development of excipients that can extend the half-lifeand/or provide stability of the peptides and proteins in the body andblood is a new area of research.

Liposomes that entrap unstable or short half-life drugs rely on thedegradation of the liposome structure before the drug can be released.Polylactic-co-glycolic acid particles are another entrapment technologythat relies on enzyme degradation of the polymer to release the drug.Semi-random grafting of two or more polymers that results in aco-polymer that binds rather than entraps the drug has been done (U.S.patent application Ser. Nos. 11/613183, 11/971482, and Castillo et al.Pharm. Res. 2012 Vol 29(1) p 306-318). Such co-polymers provide bloodstability, extension of half-life, and prolonged elevated blood level ofadministered peptides and proteins (U.S. patent application Ser. Nos.11/613183, 11/971482, and Castillo et al. Pharm. Res. 2012 Vol 29(1) p306-318).

However, the ability to improve upon the manufacturing process and thepotency of the grafted co-polymer product (the capacity to bind peptideon a per weight basis) is limited by the absence of technology that candetermine the exact organization and periodicity, if any, in which thetwo polymers arc grafted to the other polymer and relative to eachother. It appears that there is a process-induced determinant of theorganization of the components of the co-polymer molecule that thendefines the final co-polymer product composition and properties. Becausethe compositional organization of the final product cannot be evaluatedusing the existing technology, the product can only be defined by theprocesses used to manufacture said product along with theproduct-associated properties that distinguishes said product from otherproducts that are made using similar but not identical processes.Identification of such a process that makes a superior product is notobvious because of the lack of analytical technology that elucidates theatomic organization of the product and relying on the experimentation ofvarious processes and evaluating the potency of the final product cantake many years of detailed trial and error experimentation. Thedifferences in composition of the final products can only be determinedby their potency which can be defined by the process by which they aremade. This is because the polymers are large, the co-polymerizationreaction is random, and, as the reaction proceeds, the conformation ofthe polymers being grafted can change resulting in a non-randomdistribution that is determined by conformation at any given moment ofthe reaction timeline. The change in conformation is especially truewith polylysine, which is known to change from alpha helix to randomcoil to beta sheet and vice versa depending on the environment(Arunkumar et al. 1997 Int. J. Biol. Macromol. 21(3):223-230). Theconformation may also be influenced by other reagents (Mirtic andGrdadolnik 2013 Biophys. Chem. 175-176 p. 47-53) and potentially bycatalysts. These influences can remain dynamic until the reactionterminates.

Some examples of previously synthesized polymers will be describedbelow. Example 6 of U.S. patent application Ser. No. 11/613183 describespolylysine with 22% saturated with methoxypoly(ethylene glycol) (MPEG),by using MPEG succinimidyl-succinate or pre-activatedN-hydroxysuccinimidyl polyethylene glycol (NHS-PEG) to saturatepolylysine to 22%, which is materially different from the presentdisclosure (see below) that uses freshly activated MPEG-carboxyl usingNHSS (N-hydroxysuccinimidesulfate) and EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) to makesolution B, which produces polylysine saturated to 50-60%. Additionally,Example 6 of U.S. patent application Ser. No. 11/613183 modified 0.0228mmol (20 mg) primary amino equivalent, or 0.104 mmol original primaryamino based on 22% saturation of said product, by saturating theremaining primary amino with lauric acid after purification. The processof saturation used lauric acid equivalent to 2.4×mol of the originalprimary amino that was activated with NHSS equivalent to 1.1×mol of theoriginal primary amino and EDC equivalent to 5×mol of the originalprimary amino. Therefore, Example 6 of U.S. patent application Ser. No.11/613183 is a completely different process compared to the presentdisclosure (see below) based on the reagents used and their ratios. Theproduct is also a totally different product based on what can bemeasured analytically such as determination of primary amino groupsusing trinitrobenzenesulfonic acid (TNBS) giving only 22% PEGsaturation.

Example 7 of U.S. patent application Ser. No. 11/613183 describespolylysine with 22% saturated with MPEG by using MPEGsuccinimidyl-succinate or pre-activated NHS-PEG to saturate polylysineto 22% which is materially different from the present disclosure (seebelow) that uses freshly activated MPEG-carboxyl using NHSS and EDC(solution B) to produce polylysine saturated to 50-60%. Additionally,Example 7 of U.S. patent application Ser. No. 11/613183 modified 0.0228mmol (20 mg) primary amino equivalent, or 0.104 mmol original primaryamino based on 22% saturation of said product, by saturating theremaining primary amino with stearic acid after purification. Theprocess of saturation used stearic acid equivalent to 1.71×mol of theoriginal primary amino that was activated with NHSS equivalent to1.1×mol of the original primary amino and EDC equivalent to 5×mol of theoriginal primary amino. Therefore, Example 7 of U.S. patent applicationSer. No. 11/613183 is a completely different process compared to thepresent disclosure (see below) based on the reagents used and theirratios. The product is also a totally different product based on whatcan be measured analytically such as TNBS giving only 22% PEGsaturation.

Example 8 of U.S. patent application Ser. No. 11/613183 describespolylysine with 22% saturated with MPEG by using MPEGsuccinimidyl-succinate or pre-activated NHS-PEG to saturate polylysineto 22%, which is materially different from the present disclosure (seebelow) that uses freshly activated MPEG-carboxyl using NHSS and EDC(solution B) to produce polylysine saturated to 50-60%. Additionally,Example 8 of U.S. patent application Ser. No. 11/613183 modified 0.0228mmol (20 mg) primary amino equivalent, or 0.104 mmol original primaryamino based on 22% saturation of said product, by saturating theremaining primary amino with caprylic acid after purification. Theprocess of saturation used caprylic acid equivalent to 3.36×mol of theoriginal primary amino that was activated with NHSS equivalent to1.1×mol of the original primary amino and EDC equivalent to 5×mol of theoriginal primary amino. Therefore, Example 8 of U.S. patent applicationSer. No. 11/613183 is a completely different process compared to thepresent disclosure (see below) based on the reagents used and theirratios. The product is also a totally different product based on whatcan be measured analytically such as TNBS giving only 22% PEGsaturation.

Example 9 of U.S. patent application Ser. No. 11/613183 describespolylysine with 55% saturated with MPEG by using MPEGsuccinimidyl-succinate or pre-activated NHS-PEG to saturate polylysineto 55%, which is materially different from the present disclosure (seebelow) that uses freshly activated MPEG-carboxyl using NHSS and EDC(solution B) to produce polylysine saturated to 50-60%. Additionally,Example 9 of U.S. patent application Ser. No. 11/613183 modified 0.0318mmol (40 mg) primary amino equivalent of polylysine-polyethylene glycol(PLPEG), or 0.450 mmol original primary amino based on 55% saturation ofsaid PLPEG product, by saturating the remaining primary amino of saidPLPEG product with lauric acid after purification. The process ofsaturation used lauric acid equivalent to 1.8×mol of the originalprimary amino that was activated with NHSS equivalent to 0.34×mol of theoriginal primary amino and EDC equivalent to 1.16×mol of the originalprimary amino. Therefore, Example 9 of U.S. patent application Ser. No.11/613183 is a completely different process compared to the presentdisclosure (see below) based on the reagents used and their ratios. Theproduct is also a totally different product based on the presence oflauric acid or C12.

Example 12 of U.S. patent application Ser. No. 11/613183 used 1 g ofPolylysine to make solution A. MPEG-succinate (5 g, 0.59×mol equivalentof the original primary amino) was activated for 18-20 min with 250 mgNHSS (1.15 mmol or 0.68×mol equivalent of the original primary amino)and EDC (2.6 mmol or 1.53×mol equivalent of the original primary amino)to make solution B. Solution C is made by mixing solutions A and B.After 4 hours a second solution B was prepared and added to solution Cand the reaction was allowed to incubate overnight. This results in thesaturation of epsilon primary amino group of polylysine to 55%. Thefinal amounts of MPEG-succinate, NHSS, and EDC contained in solution Care 1.18×mol, 1.36×mol, and 3.06×mol equivalent of the original primaryamino respectively. Compared to the present disclosure (see below) thesesteps of the process have different ratios and timing of reagentaddition. The PLPEG product was purified, lyophilized, and the remainingprimary amino groups were saturated with stearic acid by dissolving thepurified PLPEG in 143 mL dichloromethane with 2 mmol triethylamine(0.76×mol equivalent of the original primary amino) and adding 2 mmol(0.76×mol equivalent of the original primary amino) of a freshlyactivated crude C18-NHS in dimethylformamide. The resulting product waspurified and tested for GLP-1 binding and was found to have 33% free at10% loading (see FIG. 37 of U.S. patent application Ser. No. 11/613183).When the product of the present disclosure was loaded with GLP-1 at 10%loading no free GLP-1 was observed (see Table 61 below) indicating thatthe process outlined in Example 12 of U.S. patent application Ser. No.11/613183 produced a product that is different from the presentdisclosure.

In Example 13 of U.S. patent application Ser. No. 11/613183, thepurified PLPEG (3 g) with 55% saturation of the primary amino groupsused in Example 12 of U.S. patent application Ser. No. 11/613183 wassaturated with lignoceric acid using a process similar to Example 12 ofU.S. patent application Ser. No. 11/613183. Again, the process and theproduct of this process are different from the present disclosure basedon the presence of lignoceric acid.

Examples 1-3 of U.S. patent application Ser. No. 11/971482 outlineprocesses that use pre-activated NHS-PEG thus these are differentprocesses than the present disclosure. In addition, Examples 1 and 3have 27% and 22% saturation, respectively, and therefore the product isdifferent from the process of the present disclosure. Example 2 has 55%saturation but is produced using NHS instead of NHSS, as in the presentdisclosure, so it is a different process and the product may havedifferent PEG distribution along the polylysine backbone.

Examples 4-5 of U.S. patent application Ser. No. 11/971482 used 1 g ofpolylysine with 2.4 mmol primary amine to make solution A in 200 mMHEPES. MPEG-carboxyl (5 g, 0.42×mol equivalent of the original primaryamino) was activated for 20 min with 250 mg NHSS (1.15 mmol or 0.48×molequivalent of the original primary amino) and 500 mg EDC (2.6 mmol or1.1×mol equivalent of the original primary amino) to make solution B.Activated solution B was added to solution A to make solution C. After 2hours, a second solution B was prepared and added to solution C and thereaction was allowed to incubate overnight. This results in thesaturation of epsilon primary amino groups of polylysine to 56%. Thefinal amounts of MPEG-carboxyl, NHSS, and EDC contained in solution Care 0.84×mol, 0.96×mol, and 2.2×mol equivalent of the original primaryamino respectively. Compared to the present disclosure (sec below) thesesteps of the process have different solution C final ratios, in additionto different timing of reagent addition. The PLPEG product with 56%saturation was purified and portions were saturated with behenic acidand stearic acid as described below.

Example 5 of U.S. patent application Ser. No. 11/971482 describesprocesses for behenic acid or C22 saturation, where PLPEG was made usinga process different from the present disclosure (see below); PLPEGequivalent to 1.1 of original primary amine was dissolved in 53 mLdichloromethane with 200 μL or 1.44 mmol triethylamine (1.3×molequivalent of the original primary amino) and 2.5 mmol (2.3×molequivalent of the original primary amino) of freshly activated crudeC22-NHS in 30 mL dimethylformamide:dichloromethane. This addition of C22was repeated for a second time and allowed to react overnight and theproduct purified. This process is different from the present disclosure(see below) based on reagents used and their proportions, and results ina product with behenic acid which is different from the product of thepresent disclosure (see below).

Example 5 of U.S. application Ser. No. 11/971482 describe processes forstearic acid or C18 saturation, PLPEG was made using a process differentfrom the present disclosure (see below); PLPEG equivalent to 1.1 oforiginal primary amine was dissolved in 53 mL dichloromethane with 200μL or 1.44 mmol triethylamine (1.3×mol equivalent of the originalprimary amino) and 2.5 mmol (2.3×mol equivalent of the original primaryamino) of freshly activated crude C18-NHS in 30 mLdimethylformamide:dichloromethane. This addition of C18 was repeated fora second time and allowed to react overnight and the product purified.This process is different from the present disclosure based on reagentsused and their proportions. Functionally, when loaded with GLP-1 at 2%the product of this process gives 5% free peptide (see Table 1 of U.S.patent application Ser. No. 11/971482 and Castillo et al. Pharm. Res.2012 Vol 29(1) p 306-318) whereas the present disclosure at 2% loadinggives 0% free peptide; in fact even at 5 and 10% loading the product ofthe present disclosure still shows 0% free indicating that the productof the present disclosure has a very high capacity for GLP-1 binding(see below). One is certain that the difference in properties or bindingpotency can only be explained by differences in composition.

Example 6 of U.S. patent application Ser. No. 11/971482 used 1 g ofpolylysine with 2.4 mmol primary amine to make solution A in 200 mMHEPES. MPEG-succinate (5 g, 0.42×mol equivalent of the original primaryamino) was activated for 20 min with 250 mg NHSS (1.15 mmol or 0.48×molequivalent of the original primary amino) and 500 mg EDC (2.6 mmol or1.1×mol equivalent of the original primary amino) to make solution B.Activated solution B was added to solution A to make solution C. After 2hours a second solution B was prepared and added to solution C and thereaction was allowed to incubate overnight. This results in thesaturation of epsilon primary amino group of polylysine to 57% with ahydrodynamic diameter of 14 nm. The final amounts of MPEG-carboxyl,NHSS, and EDC contained in solution C are 0.84×mol, 0.96×mol, and2.2×mol equivalent of the original primary amino respectively. Comparedto the present disclosure (see below) this process has different finalproportions in solution C as well as different timing of reagentaddition and thus the exact organization of PEG on the PL backbone mustbe different based on the properties of the final product after stearicacid saturation. The PLPEG product with 57% saturation was lyophilizedand extracted four times with 50 mL dichloromethane and saturated with2×2.5 mmol (2×mol equivalent of the original primary amino) C18-NHSdissolved in 30 mL of 1:2 vol/vol of dimethylformamide:dichloromethaneafter the addition of 400 μL or 2.88 mmol triethylamine (2.6×molequivalent of the original primary amino). This product was made using aprocess that is different from the present disclosure (see below) andproduces a product that has different potency (binding to GLP-1 at 2%loading has 5% free, see table 1 of U.S. patent application Ser. No.11/971482) compared to the product of the present disclosure (binding toGLP-1 at 2%, 5%, and 10% loading has 0% free). One is certain that thedifference in properties can only be explained by differences incomposition of the product.

Castillo et al. Pharm. Res. 2012 Vol 29(1) p 306-318 used 1 g polylysinewith 2.6 mmol primary amino dissolved in 25 ml of 1 M HEPES, pH 7.4 tomake solution A. Methoxy polyethylene glycol carboxymethyl (2 mmol or0.77×mol equivalent of the original primary amino) was dissolved in 25ml of 10 mM MES pH=4.7 with 4 mmol NHSS (1.54×mol equivalent of theoriginal primary amino), and, once dissolved, EDC (6 mmol or 2.3×molequivalent of the original primary amino) was added while stirring tomake solution B. Activation was allowed to proceed for 20 min, and theactivated MPEG-CM was added directly to the 20PL solution to makesolution C. The pH of the solution was adjusted to 7.7 using NaOH andstirred for 2 h at room temperature. An aliquot was taken, and primaryamino groups were measured by TNBS and found at 54% MPEG-CM saturationwith hydrodynamic diameter of 14.4 nm. The crude PLPEG product waslyophilized and dissolved in ˜100 ml dichloromethane and insolubleprecipitates were removed and further extracted with ˜50 mldichloromethane. The supernatants were pooled, C18-NHS (1.4×molequivalent of the original primary amino) in 20 mL dichloromethane wasadded to the pooled supernatant with magnetic stirring, thenN,N-diisopropylethylamine (DIPEA, 2.3×mol equivalent of the originalprimary amino) was added and allowed to react for 4 h. AdditionalC18-NHS (3.6 mmol or 1.4×mol equivalent of the original primary amino;with total C18-NHS added of 2.8×mol equivalent of the original primaryamino) was added and allowed to react overnight to obtain a crudeco-polymer product which was purified by ultrafiltration after solventchange to ethanol-water. This process described by Castillo et al. inPharm. Res. 2012 Vol 29(1) p 306-318 is different and has completelydifferent ratios of reagents compared to the present disclosure. Inaddition, the resulting purified co-polymer product has differentbinding properties or potency (binding to GLP-1 at 2% loading has 5%free) compared to the product of the present disclosure (binding toGLP-1 at 2%, 5%, and 10% loading has 0% free; see Table 59) indicating aunique product composition.

SUMMARY

In one aspect, the present invention provides a process of preparing asemi-random graft co-polymer comprising the steps of:

(a) dissolving a linear polyamine backbone containing W amount of freeprimary amino groups in aqueous buffer with buffering range covering pH7-8 and has a pH above 6.5 to obtain solution A having a volume Y;

(b) activating a polyethylene glycol (PEG) protective chain containing0.5-1.2×W terminal carboxyl group by mixing it with 1.7-7.0×W of NHSSand 1.5-3.6×W of EDC in aqueous buffer, pH 4-5.5 to obtain solution Bwith a final volume of Z such that W/(Y+Z)=30-55 mM and allowing theactivation to proceed for 0-30 min;

(c) mixing solution B with solution A resulting in solution C;

(d) adjusting the pH of solution C to above 6.5 if necessary;

(e) adding 0.5-1.5×W of additional EDC in small portions or all at onceto solution C after 2-3 hours and waiting until the remaining primaryamino is 55-40% of the original primary amino (45 to 60% saturation);

(f) increasing total volume of solution C when the remaining primaryamino groups is 55-40% of the original primary amino by adding 1.0-2.5volume equivalent (relative to solution C volume) of acetonitrile toobtain solution D and heating solution D to 40-70° C.;

(g) adding 0.5-6×W DIPEA or other tertiary amine to solution D;

(h) adding at least 0.75×W equivalent of C18-NHS in 40-70° C.acetonitrile to obtain solution E and stirring the solution at roomtemperature for at least 2 hours or until the remaining primary aminogroups is less than 5% of the original primary amino to obtain the crudefinal product.

In another aspect, the present invention features a semi-random graftco-polymer obtainable by the methods described herein. The semi-randomgraft co-polymer can be mixed with a peptide selected from glucagon likepeptide-1 and/or atrial natriuretic peptide, and derivatives thereof, toform a composition comprising non-covalent complex of semi-random graftco-polymer and peptide(s).

In yet another aspect, the present invention features a pharmaceuticalcomposition including a semi-random graft copolymer obtainable by themethods described herein.

In another aspect, the present invention relates to a process ofpreparing a semi-random graft co-polymer, the product of which isdifficult to fully characterize chemically. The product of the presentinvention has unique and useful properties of 1) binding to a peptideand 2) upon co-administration of the product and the peptide intoanimals the product prolongs the blood circulation time and elevates thelevel of the peptide, compared to the peptide alone without the productof the invention. The process that gives the product such propertiescannot be duplicated by any other process that is significantlydifferent from the process described in the present disclosure. Perhapsbecause of the confounding conformational changes of the backbonepolymer as chemical reactions occurs in the process of the presentdisclosure, the graft co-polymer location along the backbone is heavilydetermined by the process. The resulting product cannot be chemicallydistinguished with certainty from the product produced using otherprocesses without the challenge of developing new technology that canmonitor the process and possible conformational changes that occurduring the process. Such technology does not exist today. However, theproduct can be distinguished from the closest similar prior art productbased on its properties of 1) its superior binding to a peptide and 2)its ability to impart to said peptide an elevated concentration leveland prolonged blood circulation time upon administration into animals.The disclosure also relates to the product produced by such a processdescribed in the present specification. This product-by-process beingclaimed in the present disclosure defines a product in terms of theprocess or method (manipulative steps) used to manufacture the same. Theproduct and its known properties cannot be produced by any other knownprocess to date, despite numerous experimentations and attempts to do soas are described below. The properties of the products of U.S. patentapplication Ser. No. 11/613183 and U.S. patent application Ser. No.11/971482 were compared with those of the present disclosure. Althoughthe products of U.S. patent application Ser. No. 11/613183 and U.S.patent application Ser. No. 11/971482 bind to the same peptide as theproduct of the present disclosure, the present product has much superiorproperties in both binding to the same peptide and imparting to saidpeptide an elevated level and prolonged blood circulation time uponadministration into animals.

DETAILED DESCRIPTION

The present invention provides methods for preparing a semi-random graftco-polymer as described herein.

For example, the process of preparing a semi-random graft co-polymeroften comprises the steps of: (a) dissolving a linear polyamine backbonecontaining W amount of free primary amino groups in aqueous buffer withbuffering range covering pH 7-8 and has a pH above 6.5 to obtainsolution A having a volume Y; (b) activating a polyethylene glycol (PEG)protective chain containing 0.5-1.2×W terminal carboxyl group by mixingit with 1.7-7.0×W of NHSS and 1.5-3.6×W of EDC in aqueous buffer, pH4-5.5 to obtain solution B with a final volume of Z such thatW/(Y+Z)=30-55 mM and allowing the activation to proceed for 0-30 min;(c) mixing solution B with solution A resulting in solution C; (d)adjusting the pH of solution C to above 6.5 if necessary; (e) adding0.5-1.5×W of additional EDC in small portions or all at once to solutionC after 2-3 hours and waiting until the remaining primary amino is55-40% of the original primary amino (45 to 60% saturation); (f)increasing total volume of solution C when the remaining primary aminogroups is 55-40% of the original primary amino by adding 1.0-2.5 volumeequivalent (relative to solution C volume) of a polar organic solvent(e.g., acetonitrile) to obtain solution D and heating solution D to40-70° C.; (g) adding 0.5-6×W DIPEA or other tertiary amine to solutionD; (h) adding at least 0.75×W equivalent of C18-NHS in a polar organicsolvent (e.g., acetonitrile) at 40-70° C. to obtain solution E andstirring the solution at room temperature for at least 2 hours or untilthe remaining primary amino groups is less than 5% of the originalprimary amino to obtain the crude final product.

The methods of the present invention comprise reaction of a linearpolyamine backbone as described herein. The polyamine backbone comprisesmultiple primary amine groups as defined herein. The amounts of thedifferent reagents to be used in the reactions of the polyamine backboneare quantified by reference to the total molar amount of amine groups onthe polyamine backbone, as described herein. This total number ofpolyamine groups is denoted “W” and is given units of moles (mol).

As the skilled reader will appreciate, the amount of the reagents usedin the methods described herein may be scaled according to the amount ofprimary amine groups in the polyamine backbone. Thus, the amount of thevarious reagents used in the processes as described herein are providedin terms of multiples of “W” as defined herein, and are expressed usingthe notation “Z×W”, wherein Z is the factor by which W should bemultiplied.

The processes of the invention are scalable. Thus, the skilled personwill appreciate that the value of W is not particularly limited. Often,W will range from about 0.1 μmol to 1000 mol, more often W is from about10 μmol to about 1 mol, still more often W is from about 100 μmol toabout 100 mmol.

In the processes of the invention, the linear polyamine backbone istypically dissolved in aqueous buffer. The linear polyamine backbone ispreferably polylysine as described herein.

The aqueous buffer is not particularly limited. The buffer may be anysuitable buffer. Suitable buffers include, for example, PIPES(piperazine-N,N′-bis(2-ethanesulfonic acid)), BES ((N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid), MOPS((N-morpholino)propanesulfonic acid), HEPES (defined herein), DIPSO(N,N-Bis(2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid), TEOA(defined herein), and the like. Particularly suitable buffers includeHEPES and TEOA as defined below. Suitable buffers will typically bufferin the range pH 7-8. Typical concentrations of buffer salts in thebuffer are from 50 to 250 mM, e.g. 100 mM. For example, the buffer maybe 250 mM HEPES or 50 mM HEPES or 100 mM TEOA.

In step (a) of the process of the invention, the linear polyaminebackbone is dissolved in aqueous buffer to obtain solution A. The volumeof solution A is denoted “Y”. The pH of solution A may be adjusted usingstandard techniques, such as addition of the acid or conjugate base ofthe buffer salt, to have a desired final pH. The final pH of solution Ais typically greater than pH 6.5, such as from pH 6.5 to pH 9, e.g. fromabout pH 7 to about pH 8.

The process further comprises step (B1) or step (B2). Step B1 and StepB2 each comprise steps (b) to (d). In step B1 the process of theinvention comprises formation of a solution B as described herein, whichsolution is added to solution A to form solution C. In step B2, thereagents that are used to form solution B in step B1 are added directlyto solution A to form solution C, and solution B is not made. Thefollowing discussion applies unless otherwise stated to both steps B1and steps B2.

In step (b) of the process of the invention, a protective chain isactivated with EDC and NHSS (sulfo-NHS). The protective chain may be anysuitable polymer as described herein. Typical polymers suitable for useas the protective chain include polyethylene glycol, polypropyleneglycol and polyethylene-polypropylene glycol copolymer. The protectivechain carries a carboxyl moiety at one end of the chain. The protectivechain may or may not be alkoxylated, e.g. methoxylated or ethoxylated,at the other end of the chain. Preferred protective chains includemethoxylated polyethyleneglycol (MPEG). The average molecular mass ofthe protective chain is not particularly limited. Often, the molecularmass of the protective chain is from 2 to 20 kDa, or from 4 to 12 kDa,or more often from 4 to 6 kDa. Typically, the average molecular mass isdetermined using gel permeation technology, as described herein.

Typically, the amount of protective chain used in step (b) correspondsto 0.5×W to 1.2×W terminal carboxyl groups; more often the amount ofprotective chain corresponds to 0.5×W to 1.1×W terminal carboxyl groups,still more often from 0.5×W to 1.0×W terminal carboxyl groups. Forexample, the amount of protective chain may be from 0.85×W to 0.95×W(e.g. 0.9×W), or from 0.75×W to 0.85×W (e.g. 0.8×W), or from 0.65×W to0.75×W (e.g. 0.7×W), or from 0.55×W to 0.65×W (e.g. 0.6×W). Mosttypically, the amount of protective chain is from 0.8×W to 1×W, such asfrom 0.82×W to 0.95×W such as from 0.83×W to 0.93×W.

The protective chain is activated by reaction with EDC and NHSS.

Typically, the amount of EDC used is from 1.5×W to 3.6×W, such as from1.5×W to 3.3×W, e.g. from 1.5×W to 3.0×W. For example, the amount of EDCused in step (b) may be from 2.5×W to 2.9×W (e.g. 2.7×W), or from 2.3×Wto 2.6×W (e.g. 2.4×W), or from 2.0×W to 2.3×W (e.g. 2.1×W), or from1.7×W to 2.0×W (e.g. 1.8×W). Most typically, the amount of EDC used isfrom 2.5×W to 2.9×W, such as from 2.5×W to 2.8×W.

Typically, the amount of NHSS used is from 1.7×W to 7.0×W, such as from1.7×W to 4.0×W, e.g. from 1.7×W to 3.7×W, usually from 1.7×W to 3.4×W.For example, the amount of NHSS used in step (b) may be from 2.6×W to3.2×W (e.g. 2.7×W), or from 2.3×W to 2.8×W (e.g. 2.4×W), or from 2.0×Wto 2.5×W (e.g. 2.1×W), or from 1.7×W to 2.2×W (e.g. 1.8×W). Mosttypically, the amount of NHSS used is from 2.3×W to 2.8×W, such as from2.5×W to 2.8×W.

Thus, for example, the amount of protective chain used may be 0.5-1.2×W,the amount of EDC may be 1.5-3.6×W and the amount of NHSS may be1.7-7.0×W or 1.7-4.0×W. More typically, the amount of protective chainused may be 0.5-1.1×W, the amount of EDC may be 1.5-3.3×W and the amountof NHSS may be 1.7-3.7×W. Still more typically, the amount of protectivechain used may be 0.5-1.0×W, the amount of EDC may be 1.5-3.0×W and theamount of NHSS may be 1.7-3.4×W. Most typically, the amount ofprotective chain may be from 0.8×W to 1×W, the amount of EDC may be from2.5×W to 2.9×W and the amount of NHSS may be from 2.3×W to 2.8×W.

In step B1, the activation of the protective chain in step (b) of theprocess of the invention is conducted in aqueous buffer to yieldsolution B. The buffer which may be used is not particularly limited,and any suitable buffer can be used. Suitable buffers will typicallybuffer in the range pH 4-5.5. A suitable buffer system can include, forexample, MES (2-(N-morpholino)ethanesulfonic acid). The pH of thesolution is usually from 4.0 to 5.5, e.g. from pH 4.2 to pH 5.2, such asfrom pH 4.4 to pH 5.0, e.g. from 4.5 to 4.9. Typical concentrations ofbuffer salts in the buffer are from 1 to 100 mM, e.g., 10 mM. Forexample, the buffer may be 10 mM MES buffer, pH 4.7.

The volume of the activated protective chain in the aqueous buffersolution is denoted as volume Z. Volume Z is typically such that W/(Y+Z)is from about 30 mM to about 55 mM, e.g. from about 40 mM to about 50mM, such as about 45 mM. Volumes Y and Z are typically given in the sameunits. For example, both Y and Z are typically given in units of litres(L). The skilled person will understand that the term “litres” includesstandard variants such as μL (10⁻⁶ L), mL (10⁻³ L), cL (10⁻² L) and thelike.

The reaction time for the activation process is typically less than 30minutes, such as from 0 to 30 min, e.g. from 2 to 25 mins, often from 10to 24 mins, such as from 18 to 22 mins, e.g. about 20 mins.

In step (c) of the process of the invention, solution A as describedherein is mixed with solution B or with the reagents comprised thereinas described herein to obtain solution C. Reagents are often added undervigorous stirring in such a way that avoids precipitation. Usually,continuous stirring is used to agitate the solutions. Continuousstirring may be achieved by any suitable technique, such as by amagnetic stirrer or mixer or by manual stirring e.g. with a stirringrod. Typical stirring (rotation) speeds are from 50 to 2000 rpm (e.g.from 200 to 1500 rpm). Vigorous stirring is often required to avoidprecipitation (as described herein), and elevated rotation speeds areoften used, such as from 500 to 2000 rpm (e.g. from 1000 to 2000 rpm).Reagents and solutions are typically added slowly such as from 0 to 50mL/min, more typically from 0 to 20 ml/min (e.g., from 0 to 10 mL/min).Slow addition of solutions can be achieved using standard techniquessuch as a dropping pipette or a peristaltic pump. Other suitable methodswill be known to those skilled in the art. Slow addition of solidreagents is achieved by addition of a portion of the total reagent to beadded in any step, with time (such as from 10 s to 10 min) allowed toelapse before addition of the next portion. Alternatively, a gradualfeed of solid reagents can be achieved using, for example, a funnel orother standard techniques familiar to those skilled in the art.

Step (d) of the process of the invention is optional, and may be presentor absent. When present, step (d) corresponds to adjusting the pH ofsolution C using standard techniques, such as addition of the acid orconjugate base of the buffer salt, to have a desired final pH. The finalpH of solution C is typically greater than pH 6.5, such as from pH 6.5to pH 9, e.g. from about pH 7 to about pH 8.

In step (e) of the present invention, additional EDC is added tosolution C. The manner in which the additional EDC is added is notparticularly limited. For example, the additional EDC may be added inone administration, or multiple aliquots of EDC may be added separatelyuntil the final desired amount of EDC has been added to solution C.Thus, the additional EDC may be added in one or more portions. Theadditional EDC is typically not added to solution C immediately afterthe formation of solution C; rather, solution C is often allowed toreact for 2 to 3 hours, such as about 2.5 hours (e.g., about 2 hours)after its formation as described herein before the additional EDC isadded.

Typically, from 0.5×W to 1.5×W of additional EDC is added to solution C,more typically from 0.5×W to 1.2×W additional EDC is added, still moretypically from 0.5×W to 1.1×W, such as from 0.5×W to 1.0×W additionalEDC is added. For example, the amount of additional EDC added may befrom 0.85×W to 0.95×W (e.g. 0.9×W), or from 0.75×W to 0.85×W (e.g.0.8×W), or from 0.65×W to 0.75×W (e.g. 0.7×W), or from 0.55×W to 0.65×W(e.g. 0.6×W). Thus, the total amount of EDC in solution C in moles istypically from 2×W to 5.1×W, more typically from 2×W to 4.8×W, moretypically from 2×W to 4.4×W, still more typically from 2×W to 4×W, suchas from 3×W to 3.9×W, e.g. from about 3.4×W to about 3.8×W.

Once the additional EDC has been added, sufficient time is typicallyallowed before proceeding to any further reaction steps that may berequired for the amount of remaining primary amino groups on the linearpolyamine backbone to be from 55-40% of the original primary amino (45to 60% saturation). It is generally undesirable to freeze or lyophilizesolution C either before the additional EDC is added or shortly afteradditional EDC is added. However, solution C may be frozen orlyophilized after additional EDC has been added and the remainingprimary amino groups on the linear polyamine backbone is from 55-40% ofthe original primary amino (45 to 60% saturation). Thus, it ispreferable that solution C is not lyophilized or frozen before theadditional EDC is added or after additional EDC is added until theremaining primary amino groups on the linear polyamine backbone is from55-40% of the original primary amino (45 to 60% saturation).

The present invention also provides a method comprising steps (a) to (e)as described herein and further comprising steps (f) to (h) as describedherein.

Step (f), when present, includes obtaining solution D from solution C asdescribed herein. Step (f) may comprise:

-   -   i) freezing and lyophilizing solution C wherein the remaining        primary amino groups are 55-40% of the original primary amino        (i.e., the solution obtained from step (e) as described herein)        and reconstituting the lyophilized material in organic        solvent(s) to obtain solution D; or    -   ii) adding at least W of a strong nucleophile (such as hydroxyl        amine) to solution C wherein the remaining primary amino groups        are 55-40% of the original primary amino (i.e., the solution        obtained from step (e) as described herein) and purifying the        product by ultrafiltration, followed by lyophilization and        dissolving the product in organic solvent(s) to obtain solution        D; or    -   iii) increasing the total volume of solution C wherein the        remaining primary amino groups are 55-40% of the original        primary amino (i.e., the solution obtained from step (c) as        described herein) by adding 1.0-2.5 volume equivalents of        organic solvent(s) to obtain solution D and heating solution D        to 40-70° C., inclusive, for at least 10 minutes and adding a        strong nucleophile then purifying the product by ultrafiltration        and lyophilization; then reconstituting solution D with pure        PLPEG product in organic solvent(s) or    -   iv) increasing total volume of solution C wherein the remaining        primary amino groups are 55-40% of the original primary amino        (i.e., the solution obtained from step (e) as described herein)        by adding 1.0-2.5 volume equivalent of organic solvent(s) to        obtain solution D and heating solution D to 40-70° C.,        inclusive;

Suitable organic solvents include acetonitrile, acetone,dichloromethane, dimethylformamide, dimethyl sulfoxide and1-methyl-2-pyrrolidinone, with acetonitrile being particularly suitable.Acetonitrile as a solvent may be diluted in water or other suitablesolvents to a final concentration of less than 100% acetonitrile, forexample from 10% to 90% acetonitrile, such as from 30% to 70%acetonitrile, typically from 50% to 70% acetonitrile such as from 60% to70% acetonitrile, e.g., about 66% acetonitrile (i.e.: 66% acetonitrilein water) is often used. In some embodiments, acetonitrile can bediluted in water to a final concentration of 64%.

When step (f) is according to option (i), suitable organic solventsinclude acetonitrile, acetone, dichloromethane, dimethylformamide,dimethyl sulfoxide, and 1-methyl-2-pyrrolidinone, with acetonitrilebeing particularly suitable. The amount of solvent to be used inreconstituting the lyophilized material to obtain solution D may bereadily determined by the skilled person. Often, the amount of solventinto which the lyophilized material is reconstituted is 1.0 to 2.5volume equivalents of the volume of the solution C wherein the remainingprimary amino groups are 55-40% of the original primary amino (i.e., thesolution obtained from step (e) as described herein).

When step (f) is according to option (ii), the strong nucleophile usedis not particularly limited, and, for example, may include any strongnucleophile as described herein. Particularly suitable strongnucleophiles include NH₂OH, NaOR, LiR, NaOH or KOH, NaCN or KCN, NaCCR(acetylide anion), NaNH₂, NaNHR, NaNR₂, NaI, LiBr, KI, and NaN₃. In someembodiments, R is a C₁-C₆ alkyl group or a C₂-C₆ alkenyl group asdefined herein. When step (f) is according to option (ii),ultrafiltration can be conducted as described herein. Often, the amountof acetonitrile into which the lyophilized material is reconstituted is1.0 to 2.5 volume equivalents of the volume of the solution C whereinthe remaining primary amino groups are 55-40% of the original primaryamino (i.e., the solution obtained from step (e) as described herein).

When step (f) is according to option (iii), the solution D obtainedtherein is typically heated to from 40 to 70° C., such as from 50 to 65°C., e.g. from 55 to 60° C. Heating is typically conducted for 10-60minutes, such as from 10 to 30 minutes, e.g. 10-20 minutes. The strongnucleophile is not particularly limited, and, for example, may includeany strong nucleophile as described herein, such as the strongnucleophiles described for option (ii) of step (f) above. The amount ofacetonitrile to be used in reconstituting solution D may be readilydetermined by the skilled person. Often, the amount of acetonitrile intowhich the lyophilized material is reconstituted is 1.0 to 2.5 volumeequivalents of the volume of the solution C wherein the remainingprimary amino groups are 55-40% of the original primary amino (i.e., thesolution obtained from step (e) as described herein). For the avoidanceof doubt, when step (f) is according to option (iii), the term PLPEGproduct refers to the product of the lyophilizaton step.

When step (f) is according to option (iv), the solution D obtainedtherein is typically heated to from 40-70° C., such as from 50 to 65°C., e.g. from 55 to 60° C. Heating is typically conducted for 10 to 60minutes, such as from 10 to 30 minutes, e.g. 10 to 20 minutes.

Step (g) when present comprises adding 0.5-6×W tertiary amine tosolution D. Any suitable tertiary amine can be used, such astrimethylamine (TEA), N,N-diisopropylethylamine (DIPEA), andtriphenylamine. DIPEA is particularly suitable.

Step (h) when present comprises adding at least 0.75×W equivalent oflong chain fatty acid-NHS in 40-70° C. acetonitrile, acetone,dichloromethane, dimethylformamide, dimethyl sulfoxide, and/or1-methyl-2-pyrrolidinone to the product of step (g) to obtain solutionE, and stirring the solution at room temperature for at least 2 hours oruntil the remaining primary amino groups are less than 5% of theoriginal primary amino to obtain the crude final product.

Typically, from 0.75×W to 2×W equivalents of long chain fatty acid-NHSis added, more typically from 0.8×W to 1.5×W is added, still moretypically from 0.85×W to 1.2×W is added, such as from about 0.9×W toabout 1.1×W, e.g. about 1×W equivalent of long chain fatty acid-NHS maybe added.

The long chain fatty acid-NHS may be any suitable long chain fatty acidin which the carboxyl group is esterified with NHS. Suitable long chainfatty acids are described herein, and include those with aliphatic tailscomprising from 13 to 21 carbon atoms. Examples of long chain fattyacids which may be esterified with NHS to yield long chain fattyacid-NHS groups suitable for use in this process include those describedherein.

The long chain fatty acid-NHS is added to the product of step (g) in anorganic solvent such as a polar organic solvent, e.g. acetonitrile. Theorganic solvent is typically at 40-70° C., such as from 50 to 65° C.,e.g. from 55 to 60° C. The fatylation reaction is allowed to proceed forat least 2 hours, e.g. from 2 to 24 hours, such as from 2 hours to 12hours, e.g. from 6 to 12 hours. The reaction proceeds until theremaining primary amino groups are less than 5% of the original primaryamino, thus yielding the crude final product.

The final crude product as described herein can be purified and isolatedusing standard techniques. For example, remaining organic solvent and/orexcess fatty acids in the final product can be extracted using a solventsuch as ethyl acetate. Typically said extraction is conducted more thanonce, such as two or more times. The extracted product can then bepurified by ultrafiltration. In some embodiments, the final crudeproduct can be purified by exchanging the organic solvent into water andwashing the final product by ultrafiltration using typically at least 10volume changes of ethanol and water. The final product can be frozenand/or can be lyophilized.

In one aspect of the invention, solution B in step “b” has 0.5-1.2×Wcarboxyl group; 1.7-6.0×W of NHSS; and 1.5-3.6×W of EDC.

In another aspect of the invention, solution B in step “b” has 0.5-1.2×Wcarboxyl group; 1.7-5.0×W of NHSS; and 1.5-3.6×W of EDC.

In another aspect of the invention, solution B in step “b” has 0.5-1.2×Wcarboxyl group; 1.7-4.0×W of NHSS; and 1.5-3.6×W of EDC.

Table 1 below shows typical relationships of the reagents to each otherirrespective of the scale of the process. Table 1 refers as exemplaryaspects of the invention in steps (f), (g) and (h) to acetonitrile,DIPEA and C18-NHS, respectively; however, the skilled person willappreciate that these components are given by way of example and thatthe invention is not limited to these components, with other appropriatecomponents as described herein. Similar comments apply to Tables 2 to 12below.

TABLE 1 Step Amount/value Volume of solution a Polyamine primary aminoin W Y solution A (moles) b Protective chain carboxyl in 0.5-1.2 × W Zsolution B (moles) b NHSS in solution B (moles) 1.7-7.0 × W Z b EDC insolution B (moles) 1.5-3.6 × W Z b pH of solution B 4.0-5.5 Z bActivation time (min) 0-30 Z c & d pH of solution C above 6.5 c & dPrimary amino in solution C W/(Y + Z) = 30-55 mM (Molar) e AdditionalEDC added to solution 0.5-1.5 × W C until primary amine is 45 to[2.0-5.1 × W] 60% saturated [total EDC in C] (moles) f Acetonitrile insolution D 1.0-2.5 × vol. of solution C g DIPEA or other tertiary aminein 0.5-6 × W solution D (moles) h C18-NHS in solution E (moles) At least0.75 × W Sol D volume + 3° amine + C18-NHS volume

Often, the linear polyamine backbone is polylysine having a degree ofpolymerization of 35-150 based on light scattering or nuclear magneticresonance (NMR) analysis. Usually, the linear polyamine backbone ispolylysine with degree of polymerization of 35-85 based on lightscattering or nuclear magnetic resonance (NMR) analysis.

Typically, the PEG protective chain is methoxy PEG (MPEG) having asingle carboxyl terminus and has 4-12 kDa number average molecularweight or Mn based on Gel Permeation Chromatography (GPC). Often, thePEG protective chain is methoxy PEG chain with a single carboxylterminus and has 4-6 kDa number average molecular weight or Mn based onGel Permeation Chromatography (GPC).

In another aspect of the present invention, the above aforementionedprocess is such wherein in step “e”, 0.5-1.2×W of EDC is added tosolution C; wherein in step “f”, 1.5-2.0 volume equivalent of a polarorganic solvent (e.g., acetonitrile) is added when the remaining primaryamino groups reaches 55-40% of the original primary amino. Table 2 belowshows the relationship of the reagents to each other in the process justdescribed irrespective of the scale of the process.

TABLE 2 Step Amount/value Volume of solution a Polyamine primary aminoin W Y solution A (moles) b Protective chain carboxyl in 0.5-1.2 × W Zsolution B (moles) b NHSS in solution B (moles) 1.7-4.0 × W Z b EDC insolution B (moles) 1.5-3.6 × W Z b pH of solution B 4.0-5.5 Z bActivation time (min) 0-30 c & d pH of solution C above 6.5 c & dPrimary amino in solution C W/(Y + Z) = 30-55 mM (Molar) e AdditionalEDC added to 0.5-1.2 × W solution C until primary amine [2.0-4.8 × W] is45 to 60% saturated [total EDC in C] (moles) f Acetonitrile in solutionD 1.5-2.0 x vol. of solution C g DIPEA or other tertiary amine 0.5-6 × Win solution D (moles) h C18-NHS in solution E (moles) At least 0.75 × WSol D volume + 3° amine + C18-NHS volume

In another aspect of the present invention, the above aforementionedprocess is such wherein solution B in step “b” has 0.5-1.1×W carboxylgroup; 1.7-3.7×W of NHSS; and 1.5-3.3×W of EDC; wherein in step “e”,0.5-1.1×W of EDC is added to solution C. Table 3 below shows therelationship of the reagents to each other in the process just describedirrespective of the scale of the process.

TABLE 3 Step Amount/value Volume of solution a Primary amino in solutionA W Y (moles) b Carboxyl in solution B (moles) 0.5-1.1 × W Z b NHSS insolution B (moles) 1.7-3.7 × W Z b EDC in solution B (moles) 1.5-3.3 × WZ b pH of solution B 4.0-5.5 Z b Activation time (min) 0-30 c & d pH ofsolution C above 6.5 c & d Primary amino in solution C W/(Y + Z) = 30-55mM (Molar) e Additional EDC added to 0.5-1.1 × W solution C after 3hours [total [2.0-4.4 × W] EDC in C] (moles) f Acetonitrile in solutionD 1.5-2.0 volume of solution C g DIPEA or other tertiary amine 0.5-6 × Win solution D (moles) h C18-NHS in solution E (moles) At least 0.75 × WSol D volume + 3° amine + C18-NHS volume

In another aspect of the present invention, the above aforementionedprocess is such wherein solution B in step “b” has 0.5-1.0×W carboxylgroup; 1.7-3.4×W of NHSS; 1.5-3.0×W of EDC; wherein activation isallowed to proceed for 5-25 min; wherein in step “e”, 0.5-1.0×Wadditional EDC is added to solution C. Table 4 below shows therelationship of the reagents to each other in the process just describedirrespective of the scale of the process.

TABLE 4 Step Amount/value Volume of solution A Primary amino in solutionA W Y (moles) B Carboxyl in solution B (moles) 0.5-1.0 × W Z B NHSS insolution B (moles) 1.7-3.4 × W Z B EDC in solution B (moles) 1.5-3.0 × WZ B pH of solution B 4.0-5.5 Z B Activation time (min) 5-25 c & d pH ofsolution C above 6.5 c & d Primary amino in solution C W/(Y + Z) = 30-55mM (Molar) E Additional EDC added to solution 0.5-1.0 × W [2.0-4.0 × W]C [total EDC in C] (moles) F Acetonitrile in solution D 1.5-2.0 volumeof solution C G DIPEA or other tertiary amine in 0.5-6 × W solution D(moles) H C18-NHS (moles) At least 0.75 × W Sol D volume + DIPEA +C18-NHS volume

In another aspect of the present invention, the above aforementionedprocess is such wherein solution B in step “b” has 0.85-0.95×W carboxylgroup; 2.6-3.2×W of NHSS; 2.5-2.9×W of EDC; wherein the pH of solution Bis 4.4-5.0; wherein activation is allowed to proceed for 18-22 min;wherein in step “e”, 0.85-0.95×W of EDC is added to solution C; whereinthe pH of solution C is adjusted to 7-8; wherein in step “g”, 1-3×WDIPEA or other tertiary amine is added to solution D. Table 5 belowshows the relationship of the reagents to each other in the process justdescribed irrespective of the scale of the process.

TABLE 5 Step Amount/value Volume of solution a Primary amino in solutionA W Y (moles) b Carboxyl in solution B (moles) 0.85-0.95 × W Z b NHSS insolution B (moles) 2.6-3.2 × W Z b EDC in solution B (moles) 2.5-2.9 × WZ b pH of solution B 4.4-5.0 Z b Activation time (min) 18-22 c & d pH ofsolution C 7-8 c & d Primary amino in solution C W/(Y + Z) = 30-55 mM(Molar) e Additional EDC added to 0.85-0.95 × W [3.4-3.9 × W] solution C[total EDC in C] (moles) f Acetonitrile in solution D 1.5-2.0 volume ofsolution C g DIPEA or other tertiary amine 1-3 × W in solution D (moles)h C18-NHS (moles) At least 0.75 × W Sol D volume + DIPEA + C18-NHSvolume

In another aspect of the present invention, the above aforementionedprocess is such wherein solution B in step “b” has 0.9×W carboxyl group;2.7×W of NHSS; 2.7×W of EDC; wherein the pH of solution b is 4.4-5.0;wherein activation is allowed to proceed for 18-22 min; wherein the pHof solution C is adjusted to 7-8; wherein in step “e”, 0.9×W additionalEDC is added to solution C. Table 6 below shows the relationship of thereagents to each other in the process just described irrespective of thescale of the process.

TABLE 6 Step Amount/value Volume of solution a Primary amino in solutionA W Y (moles) b Carboxyl in solution B (moles) 0.9 × W Z b NHSS insolution B (moles) 2.7 × W Z b EDC in solution B (moles) 2.7 × W Z b pHof solution B 4.4-5.0 Z b Activation time (min) 18-22 c & d pH ofsolution C 7-8 c & d Primary amino in solution C W/(Y + Z) = 30-55 mM(Molar) e Additional EDC added to 0.9 × W [3.6 × W] solution C [totalEDC in C] (moles) f Acetonitrile in solution D 1.5-2.0 volume ofsolution C g DIPEA or other tertiary amine in 1-3 × W solution D (moles)h C18-NHS (moles) At least 0.75 × W Sol D volume + 3° amine + C18-NHSvolume

In another aspect of the present invention, the above aforementionedprocess is such wherein solution B in step “b” has 0.75-0.85×W carboxylgroup, 2.3-2.8×W of NHSS, and 2.3-2.6×W of EDC; wherein the pH ofsolution B is 4.4-5.0; wherein activation is allowed to proceed for18-22 min; wherein the pH of solution C is adjusted to 7-8; wherein instep “e”, 0.75-0.85×W of EDC is added to solution C; wherein in step“g”, 1-3×W DIPEA or other tertiary amine is added to solution D. Table 7below shows the relationship of the reagents to each other in theprocess just described irrespective of the scale of the process.

TABLE 7 Step Amount/value Volume of solution a Primary amino in solutionA W Y (moles) b Carboxyl in solution B (moles) 0.75-0.85 × W Z b NHSS insolution B (moles) 2.3-2.8 × W Z b EDC in solution B (moles) 2.3-2.6 × WZ b pH of solution B 4.4-5.0 Z b Activation time (min) 18-22 c & d pH ofsolution C 7-8 c & d Primary amino in solution C W/(Y + Z) = 30-55 mM(Molar) e Additional EDC added to 0.75-0.85 × W solution C [total EDC inC] [3.1-3.5 × W] (moles) f Acetonitrile in solution D 1.5-2.0 volume ofsolution C g DIPEA or other tertiary amine in 1-3 × W solution D (moles)h C18-NHS (moles) At least 0.75 × W Sol D volume + 3° amine + C18-NHSvolume

In another aspect of the present invention, the above aforementionedprocess is such wherein solution B in step “b” has 0.8×W carboxyl group;2.4×W of NHSS; 2.4×W of EDC; wherein the pH of solution B is 4.4-5.0;wherein activation is allowed to proceed for 18-22 min; wherein the pHof solution C is adjusted to 7-8; wherein in step “e”, 0.8×W additionalEDC is added to solution C; wherein in step “e” at least 0.75×W C18-NHSis added. Table 8 below shows the relationship of the reagents to eachother in the process just described irrespective of the scale of theprocess.

TABLE 8 Step Amount/value Volume of solution a Primary amino in solutionA W Y (moles) b Carboxyl in solution B (moles) 0.8 × W Z b NHSS insolution B (moles) 2.4 × W Z b EDC in solution B (moles) 2.4 × W Z b pHof solution B 4.4-5.0 Z b Activation time (min) 18-22 c & d pH ofsolution C 7-8 c & d Primary amino in solution C W/(Y + Z) = 30-55 mM(Molar) e Additional EDC added to 0.8 × W [3.2 × W] solution C [totalEDC in C] (moles) f Acetonitrile in solution D 1.5-2.0 volume ofsolution C g DIPEA or other tertiary amine in 1-3 × W solution D (moles)h C18-NHS (moles) At least 0.75 × W Sol D volume + 3° amine + C18-NHSvolume

In another aspect of the present invention, the above aforementionedprocess is such wherein solution B in step “b” has 0.65-0.75×W carboxylgroup, 2.0-2.5×W of NHSS, 2.0-2.3×W of EDC; wherein the pH of solution Bis 4.4-5.0; wherein activation is allowed to proceed for 18-22 min;wherein the pH of solution C is adjusted to 7-8; wherein in step “e”,0.65-0.75×W of EDC is added to solution C; wherein in step “g”, 1-3×WDIPEA or other tertiary amine is added to solution D. Table 9 belowshows the relationship of the reagents to each other in the process justdescribed irrespective of the scale of the process.

TABLE 9 Step Amount/value Volume of solution a Primary amino in solutionA W Y (moles) b Carboxyl in solution B (moles) 0.65-0.75 × W Z b NHSS insolution B (moles) 2.0-2.5 × W Z b EDC in solution B (moles) 2.0-2.3 × WZ b pH of solution B 4.4-5.0 Z b Activation time (min) 18-22 c & d pH ofsolution C 7-8 c & d Primary amino in solution C W/(Y + Z) = 30-55 mM(Molar) e Additional EDC added to 0.65-0.75 × W [2.7-3.1 × W] solution C[total EDC in C] (moles) f Acetonitrile in solution D 1.5-2.0 volume ofsolution C g DIPEA or other tertiary amine 1-3 × W in solution D (moles)h C18-NHS (moles) At least 0.75 × W Sol D volume + 3° amine + C18-NHSvolume

In another aspect of the present invention, the above aforementionedprocess is such wherein solution B in step “b” has 0.7×W carboxyl group;2.1×W of NHSS; 2.1×W of EDC; wherein the pH of solution B is 4.4-5.0;wherein activation is allowed to proceed for 18-22 min; wherein the pHof solution C is adjusted to 7-8; wherein in step “e”, 0.7×W additionalEDC is added to solution C; wherein in step “g”, 1-3×W DIPEA or othertertiary amine is added to solution D. Table 10 below shows therelationship of the reagents to each other in the process just describedirrespective of the scale of the process.

TABLE 10 Step Amount/value Volume of solution a Primary amino insolution A W Y (moles) b Carboxyl in solution B (moles) 0.7 × W Z b NHSSin solution B (moles) 2.1 × W Z b EDC in solution B (moles) 2.1 × W Z bpH of solution B 4.4-5.0 Z b Activation time (min) 18-22 c & d pH ofsolution C 7-8 c & d Primary amino in solution C W/(Y + Z) = 30-55 mM(Molar) e Additional EDC added to 0.7 × W [2.8 × W] solution C [totalEDC in C] (moles) f Acetonitrile in solution D 1.5-2.0 volume ofsolution C g DIPEA or other Tertiary amine 1-3 × W in solution D (moles)h C18-NHS (moles) At least 0.75 × W Sol D volume + 3° amine + C18-NHSvolume

In another aspect of the present invention, the above aforementionedprocess is such wherein solution B in step “b” has 0.55-0.65×W carboxylgroup, 1.7-2.2×W of NHSS, 1.7-2.0×W of EDC; wherein the pH of solution Bis 4.4-5.0; wherein activation is allowed to proceed for 18-22 min;wherein the pH of solution C is adjusted to 7-8; wherein in step e,0.55-0.65×W of EDC is added to solution C; wherein in step “g”, 1-3×WDIPEA or other tertiary amine is added to solution D. Table 11 belowshows the relationship of the reagents to each other in the process justdescribed irrespective of the scale of the process.

TABLE 11 Step Amount/value Volume of solution a Primary amino insolution A W Y (moles) b Carboxyl in solution B (moles) 0.55-0.65 × W Zb NHSS in solution B (moles) 1.7-2.2 × W Z b EDC in solution B (moles)1.7-2.0 × W Z b pH of solution B 4.4-5.0 Z b Activation time (min) 18-22c & d pH of solution C 7-8 c & d Primary amino in solution C W/(Y + Z) =30-55 mM (Molar) e Additional EDC added to 0.55-0.65 × W solution C[total EDC in C] [2.3-2.7 × W] (moles) f Acetonitrile in solution D1.5-2.0 volume of solution C g DIPEA or other tertiary amine 1-3 × W insolution D (moles) h C18-NHS (moles) At least 0.75 × W Sol D volume + 3°amine + C18-NHS volume

In another aspect of the present invention, the above aforementionedprocess is such wherein solution B in step “b” has 0.6×W carboxyl group;1.8×W of NHSS; 1.8×W of EDC; wherein the pH of solution B is 4.4-5.0;wherein activation is allowed to proceed for 18-22 min; wherein the pHof solution C is adjusted to 7-8; wherein in step “e”, 0.6×W additionalEDC is added to solution C; wherein in step “e” at least 0.75×W C18-NHSis added. Table 12 below shows the relationship of the reagents in theprocess just described to each other irrespective of the scale of theprocess.

TABLE 12 Step Amount/value Volume of solution a Primary amino insolution A W Y (moles) b Carboxyl in solution B (moles) 0.6 × W Z b NHSSin solution B (moles) 1.8 × W Z b EDC in solution B (moles) 1.8 × W Z bpH of solution B 4.4-5.0 Z b Activation time (min) 18-22 c & d pH ofsolution C 7-8 c & d Primary amino in solution C W/(Y + Z) = 30-55 mM(Molar) e Additional EDC added to 0.6 × W solution C [total EDC in C][2.4 × W] (moles) f Acetonitrile in solution D 1.5-2.0 volume ofsolution C g DIPEA or other tertiary amine 1-3 × W in solution D (moles)h C18-NHS (moles) At least 0.75 × W Sol D volume + 3° amine + C18-NHSvolume

As described herein, the present invention includes alternatives withinsteps (f), (g), and (h) of the above aforementioned processes, wherein:(f) comprises freezing and lyophilizing solution C when the remainingprimary amino groups is 55-40% of the original primary amino andreconstituting the lyophilized material with water-immiscible organicsolvent such as dichloromethane or a mixture of water-miscible andwater-immiscible organic solvents to obtain solution D; (g) comprisesadding 0.5-6×W DIPEA or other tertiary amine to solution D; (h)comprises adding at least 0.75×W equivalent of C18-NHS directly or inappropriate solvent such as dichloromethane to obtain solution E andstirring the solution at room temperature for at least 2 hours until theremaining primary amino groups is less than 5% of the original primaryamino to obtain the crude final product.

The above aforementioned processes, wherein the final product insolution E is in organic solvent, may further comprise step (i)exchanging the organic solvent in the crude product into water andwashing the product by ultrafiltration using at least 10 exchanges ofethanol and water.

As described herein, the present invention includes further alternativeswithin steps (f), (g), and (h) of above aforementioned processes,wherein: (f) comprises adding a strong nucleophile (at least 1×Wequivalent of the original primary amino) such as hydroxyl amine tosolution C and purifying the product by ultrafiltration as describedbelow followed by lyophilization and dissolving in a polar organicsolvent (e.g., acetonitrile) to obtain solution D; (g) comprises adding0.5-6×W DIPEA or other tertiary amine to solution D; (h) comprisesadding at least 0.75×W equivalent of C18-NHS to obtain solution E andstirring the solution at room temperature for at least 2 hours or untilthe remaining primary amino groups is less than 5% of the originalprimary amino to obtain the crude final product.

The above aforementioned processes, wherein the final product insolution E is in a mixture of water and water-miscible organic solvent,may further comprise step (i) extracting the water-miscible organicsolvent and excess fatty acids from the crude final product in solutionE using a polar organic solvent (e.g., ethyl acetate) and discarding thepolar organic solvent (e.g., ethyl acetate) layer and repeating theextraction of the water layer at least once followed by washing theproduct by ultrafiltration using at least 10 exchanges of ethanol andwater.

Definitions

For convenience, before further description of the present disclosure,certain terms employed in the specification, examples, and appendedclaims are collected here. These definitions should be read in light ofthe remainder of the disclosure and understood as by a person ofordinary skill in the art. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by a person of ordinary skill in the art. Unless otherwiseindicated the general mathematical rule of rounding off numbers appliesto all the numbers in the present specification with the exception ofmolecular weight of a non-poly-disperse molecule such as for exampleH₂O, NHSS, EDC etc. Polymers are poly-disperse molecules. Whenever anumber is given the last digit of the number is understood to be thelimit of certainty and is a result of rounding off the range of numbersto the nearest last digit of a given number. For example the “5 kDapolymer” means a range between 4.5 kDa to 5.5 kDa since rounding of4.51-5.49 kDa to the nearest thousand is 5 kDa. Another example is 2.1mmol is a range between of 2.05 to 2.15 mmol. Another example is 5.0 kDapolymer is a range between 4.95 to 5.05 kDa.

The articles “a” and “an” are used to refer to one or to more than one(i.e., to at least one) of the grammatical object of the article. By wayof example, “a protective chain” means one protective chain or more thanone protective chain.

The term “ANP” is Atrial Natriuretic Peptide (sequence based onconvention known in the art: SEQ ID NO: 1-SLRRSSCFGGRMDRIGAQSGLGCNSFRYwith intrapeptide disulfide bond). This peptide was used for the purposeof the present specification to distinguish the differences betweengraft co-polymers produced by various processes.

The term “BNP” is a B type Natriuretic Peptide (sequence based onconvention known in the art: SEQ ID NO: 2-SPKMVQGSGCFGR KMDRISSSSGLGCKVLRRH with intrapeptide disulfide bond). This peptide was used forthe purpose of the present specification to distinguish the differencesbetween graft co-polymers produced by various processes.

The terms “C12-NHS”, “C13-NHS”, “C14-NHS”, “C15-NHS”, “C16-NHS”,“C17-NHS”, “C18-NHS”, “C19-NHS”, “C20-NHS”, “C21-NHS”, etc. refer tofatty acids with aliphatic tails of 12 to 21 carbons in which thecarboxyl group is esterified with NHS, see “fatty acids” below. Unlessotherwise stated, C12 refers to lauric acid, C14 refers to myristicacid, C16 refers to palmitic acid, C18 refers to stearic acid, and C20refers to arachidic acid.

The term “derivative” or “analog” as used herein refers to a compoundwhose core structure is the same as, or closely resembles that of, aparent compound but which has a chemical or physical modification suchas different or additional groups. The term also includes a peptide withat least 80% sequence identity (i.e. amino acid substitution is lessthan 20%) with the parent peptide. The term also includes a peptide withadditional groups attached to it compared to the parent peptide, such asfatty acids and/or additional amino acids. The term also includes apolymer with additional group(s) attached to it, such as, in the case ofa protective group, an alkoxy group, compared to the parent polymer. Theterm also includes methoxylated or ethoxylated protective chains withadditional methoxy or ethoxy-group(s) attached to it, compared to theparent protective chains.

The term “DIPEA” refers to N,N-Diisopropylethylamine, or Hünig's base,or DIEA, an organic compound and an amine. It is used in organicchemistry as a base. Because the nitrogen atom is shielded by the twoisopropyl groups and an ethyl group, only a proton is small enough toeasily fit. Like 2,2,6,6-tetramethylpiperidine, this compound is a goodbase but a poor nucleophile, which makes it a useful organic reagent.

The term “EDC” refers to 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimideHCl which can also be referred to as EDAC or EDCI and has a MolecularWeight: 191.70 Da. This carbodiimide reagent contains a functional groupconsisting of the formula N═C═N which is necessary for activation ofcarboxyl groups; this group is important for activating carboxyl groupson the protective chain and the fatty acids. During the process of thecoupling reactions, the activated carboxyl groupO-acylisourea-intermediate can be stabilized by formingN-hydroxysuccinimide ester with either N-hydroxysuccinimide orN-hydroxysulfosuccinimide.

The term “fatty acid” is a carboxylic acid with a long aliphatic tail orchain), which is either saturated or unsaturated. Most naturallyoccurring fatty acids have a tail of an even number of carbon atoms,from 4 to 28. When they arc not attached to other molecules, they areknown as “free” fatty acids. Fatty acids that have carbon-carbon doublebonds are known as unsaturated and those without double bonds are knownas saturated. Fatty acid chains differ by length, often categorized asshort to very long. Short-chain fatty acids are fatty acids withaliphatic tails of fewer than six carbons (i.e. butyric acid).Medium-chain fatty acids are fatty acids with aliphatic tails of 6 to 12carbons (also called C6-C12 fatty acids, where the number refers to thenumber of carbons). Long-chain fatty acids are fatty acids withaliphatic tails 13 to 21 carbons (also called C13-C21 fatty acids). Verylong chain fatty acids are fatty acids with aliphatic tails longer than22 carbons (also called ≥C22 fatty acids). For the purpose of thepresent specification, the term “fatty acid” covers all of the above andeach of the species of fatty acid may have more than one common name.Examples of saturated fatty acids includes caprylic acid(CH₃(CH₂)₆COOH), capric acid (CH₃(CH₂)₈COOH), lauric acid(CH₃(CH₂)₁₀COOH), myristic acid (CH₃(CH₂)₁₂COOH), palmitic acid(CH₃(CH₂)₁₄COOH), stearic acid (CH₃(CH₂)₁₆COOH), arachidic acid(CH₃(CH₂)₁₈COOH), behenic acid (CH₃(CH₂)₂₀COOH), lignoceric acid(CH₃(CH₂)₂₂COOH), cerotic acid (CH₃(CH₂)₂₄COOH). Examples of unsaturatedfatty acids includes myristoleic acid (CH₃(CH₂)₃CH═CH(CH₂)₇COOH),palmitoleic acid (CH₃(CH₂)₅CH═CH(CH₂)₇COOH), sapienic acid(CH₃(CH₂)₈CH═CH(CH₂)₄COOH), oleic acid (CH₃(CH₂)₇CH═CH(CH₂)₇COOH),elaidic acid (CH₃(CH₂)₇CH═CH(CH₂)₇COOH), vaccenic acid(CH₃(CH₂)₅CH═CH(CH₂)₉COOH), linoleic acid(CH₃(CH₂)₄CH═CHCH₂CH═CH(CH₂)₇COOH), linoelaidic acid(CH₃(CH₂)₄CH═CHCH₂CH═CH(CH₂)₇COOH), α-linolenic acid(CH₃CH₂CH═CHCH₂CH═CHCH₂CH═CH(CH₂)₇COOH), arachidonic acid(CH₃(CH₂)₄CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CH(CH₂)₃COOH), eicosapentaenoicacid (CH₃CH₂CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CH(CH₂)₃COOH), erucicacid (CH₃(CH₂)₇CH═CH(CH₂)₁₁COOH), docosahexaenoic acid(CH₃CH₂CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CH(CH₂)₂ COOH).

The term “fatty acid-NHS” refers to the fatty acid in which the carboxylgroup is esterified with NHS. The term “long chain fatty acid-NHS”refers to the long-chain fatty acids or fatty acids with aliphatic tails13 to 21 carbons (also called C13-C21 fatty acids, where the numberrefers to the number of carbons) in which the carboxyl group isesterified with NHS. Similar naming applies to “short long chain fattyacid-NHS” where the fatty acids have aliphatic tails of 6 to 12 carbons.Again similar naming applies to “very long chain fatty acid-NHS” wherethe fatty acids have aliphatic tails of 22 carbons and greater.

The term “Fatylation” refers to a step or a chemical process of adding afatty acid to a polyamine backbone.

The term “GLP-1” refers to glucagon like peptide-1 (sequence SEQ ID NO:3-HAEGTFTSDV SSYLEGQAAK EFIAWLVKGR G). This peptide was used for thepurpose of the present specification to distinguish the differencesbetween graft co-polymers produced by various processes.

The term “HEPES” refers to 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid and is a zwitterionic organic chemical buffering agent with highbuffering capacity between pH 6.5 and 8.5.

The term “linear polyamine backbone” refers to a straight chainnon-proteinaceous homo- or hetero-polymer with repeating primary aminogroups and may be of natural or synthetic origin. Non-proteinaceousmeaning that it is not a protein made by a living organism to have athree dimensional conformation associated with cellular activity. The“linear polyamine backbone” is used interchangeably with “linearpolymeric backbone” and is a component of solution A in thisspecification. The preferred straight “linear polyamine backbone” ispolylysine and is also referred to as PL in this specification. The“linear polyamine backbone” can be other polyamino acids which may haveD- or L-chirality, or both, in which the side group of the amino acid(known as the R-group) contains a primary amine. Often, the polymericbackbone may have an average number molecular weight (Mn) of 5-95 kDa ora degree of polymerization (DPn) of 22-450 based on light scattering ornuclear magnetic resonance analysis. The preferred polymeric backbonehas a Mn of 7-32 kDa or a DPn of 35-150 based on light scattering ornuclear magnetic resonance analysis. The most preferred polymericbackbone has a Mn of 7-18 kDa or a DPn of 35-85 based on lightscattering or nuclear magnetic resonance analysis. By viscositymeasurement the DPns given above can be twice as high for the samematerial depending on what standard is used. Other polymeric backboneswith repeating primary amino groups may also be used such carbohydratepolymers or other synthetic polymers. The polymeric backbone providesthe multiple primary amino groups to which the protective chains andfatty acids can be attached.

The term “% loading” is defined as a binding characteristic wherepeptide is mixed with the semi-random graft co-polymer product at adefined weight-to-weight ratio of peptide:co-polymer in a physiologicalor near physiological buffer, for example 10% loading consists of 1 partpeptide and 10 parts co-polymer, by weight. The percentage of freepeptide at particular loadings is used to distinguish differencesbetween graft co-polymers produced by various processes.

The term “protective side chain” as used herein refers to a hydrophilicpolymer molecule that has the ability to extensively bind or imbibewater along the chain and is a component of solution B. Bind or imbibeis different from absorb which is refers to uptake into the spaces orchannels of a large molecular structure such as sponge, a resin, or gel.Because of this extensive binding with water molecules, the protectivechain has high water solubility and it also increases the watersolubility of other polymers to which it is linked. The protective sidechain does not have a significant amount of charge and is generallynon-immunogenic. This also means that the protective chain provides ahydrophilic property to the composition that would otherwise be lesswater soluble. The term “protective chain” and “protective side chain”are used in this specification interchangeably. The protective chains ofthe present composition typically include polyoxyethylene glycol, alsoreferred to as polyethylene glycol, which may or may not be alkoxylated(such as methoxy or ethoxy) at one end but terminates with a carboxylmoiety at the other end. The protective chains may also or alternativelybe polypropylene glycol or polyethylene-polypropylene glycol co-polymer,which may or may not be alkoxylated (such as methoxy or ethoxy) at oneend but all terminate with a carboxyl moiety. The preferred protectivechain is a linear methoxylated polyethyleneglycol (MPEG or methoxyPEG)with carboxyl terminus and which ranges in size from 2 to 20 kDa basedon gel permeation chromatography standardized with the same or similarmaterials. The more preferred size is 4-12 kDa and the most preferredsize is 4-6 kDa. The protective chains of the present composition alsoinclude uncharged polysaccharides and their derivatives such asethoxylated or methoxylated polysaccharides. In this context, unchargedmeans that the main body of the chain does not have positive or negativecharge.

The term “MPEG-CM” refers to methoxypolyethyleneglycol-carboxyl and is alinear PEG with methoxy group at one end and a carboxyl group at theother end. This is a non-pre-activated form of PEG.

The term “MPEG-SCM” refers to methoxypolyethyleneglycol succinimidylsuccinate and is an NHS pre-activated form of linear PEG with a methoxygroup at one end and an NHS linked carbonyl group at the other end.

The term “NHS” refers to N-hydroxysuccinimide and has a molecularweight: 115.10 Da.

The term “NHSS” refers to N-hydroxysulfosuccinimide and has a molecularweight: 217.14 Da.

The term “non-water miscible organic solvent” means that the organicsolvent mixes with water or dissolves in water at less than 50% wt/wt.For example: dichloroethane, benzene, butyl acetate, carbontetrachloride, chlorobenzene, chloroform, Cyclohexane, diethyl ether,di-isopropylether, dichloromethane, ethyl acetate, ethylbenzene, methylethyl ketone, methyl butyl ether, n-butanol, pentane, n-hexane, heptane,toluene, tetrachloroethylene, and xylene.

The term “PEGylation” refers to a step or a chemical process of addingPEG or its derivative to a polyamine backbone.

The term “primary amines” refers to a nitrogen bonded to one alkyl oraromatic ring and 2 hydrogen atoms. In other words, it is a nitrogenwhere one of the three hydrogen sites is replaced by an organicsubstituent. Important primary amines for the purpose of thisspecification are the repeating primary amine moieties along the linearpolyamine backbone. In the case of polylysine, it is the epsilon primaryamino group of lysine along the polylysine polymer.

The term “Sol” refers to solution and for the purpose of the presentspecification refers to various liquid solutions A through E designatedSol A to Sol E.

The term “strong nucleophiles” refers to reagents that get easilydeprotonated to give anions with a full negative charge, are easilyrecognizable by the presence of sodium, lithium, or potassiumcounterions, and participate in SN2-type substitutions. Examples ofstrong nucleophile include NH₂OH, NaOCH₃ (any NaOR), LiCH₃ (any LiR),NaOH or KOH, NaCN or KCN, NaCCR (acetylide anion), NaNH₂, NaNHR, NaNR₂,NaI, LiBr, KI, and NaN₃. A strong nucleophile can be used to stop thereaction in Sol C of the present disclosure to interrupt the process ata specific stage for purification and/or evaluation of intermediate.

The term “TEA” refers to triethylamine is the chemical compound with theformula N(CH₂CH₃)₃, commonly abbreviated Et₃N. It is also abbreviatedTEA, yet this abbreviation must be used carefully to avoid confusionwith triethanolamine or tetraethylammonium, for which TEA is also acommon abbreviation. Like diisopropylethylamine (Hünig's base),triethylamine is commonly encountered in organic synthesis.

The term “TEOA” refers to triethanolamine or2,2′,2″-trihydroxy-triethylamine or tris(2-hydroxyethyl) amine, alsoabbreviated as TEA, is a viscous organic compound that is both atertiary amine and a triol.

The term “tertiary amines” refers to a nitrogen where the three hydrogensites are replaced by three organic substituents. Examples includetrimethylamine (TEA), N,N-diisopropylethylamine (DIPEA), andtriphenylamine.

The term “TNBS” refers to Trinitrobenzenesulfonic acid (C₆H₃N₃O₉S) whichis a nitro-aryl oxidizing acid. For the purpose of this specification,an assay utilizing TNBS is used to measure primary amine according toSpadaro, A. C., et al., (A convenient manual trinitrobenzenesulfonicacid method for monitoring amino acids and peptides in chromatographiccolumn effluents. (Anal. Biochem., 1979, 96(2): p. 317-21) in solutionsA, C, and E. The use of the TNBS assay for primary amine is to determinethe primary amino group saturation of solution C so the reaction can goto the next step.

The term “water-miscible organic solvent” means that the organic solventmixes with water or dissolves in water at 50% wt/wt or greater, forexample acetic acid, acetonitrile, acetone, dimethyl formamide, dimethylsulfoxide, dioxane, ethanol, isopropanol, n-propanol, methanol, andtetrahydrofuran.

The term “W” refers to the total number of moles of primary amino grouppresent in the starting polyamine backbone in solution A or originalamino groups in solution A of the process. The use of the term allowsfor the process to be increased to industrial scale. For the purpose ofclarity, critical reagents in this specification are expressed asfractions or multiples of “W”.

The term “×W” refers to multiples of W and is usually preceded by anumber that will be multiplied by W to give a specific value or amountof critical reagent needed. For example if W is equal to 2.6 mmol and aprotective chain has a carboxyl equivalent of 0.9×W, then that amountwill be 0.9'2.6 mmol, or 2.34 mmol.

As used herein, a C₁ to C₆ alkyl group is a linear or branched alkylgroup containing from 1 to 6 carbon atoms. Typically a C₁ to C₆ alkylgroup is a C₁ to C₄ alkyl group, which is a linear or branched alkylgroup containing from 1 to 4 carbon atoms. A C₁ to C₄ alkyl group isoften a C₁ to C₃ alkyl group. Examples of C₁ to C₆ alkyl groups includemethyl, ethyl, n-propyl , iso-propyl, n-butyl, sec-butyl, tert-butyl,pentyl and hexyl. A C₁ to C₃ alkyl group is typically a C₁ to C₂ alkylgroup. A C₁ to C₂ alkyl group is methyl or ethyl, typically methyl. Forthe avoidance of doubt, where two alkyl groups are present, the alkylgroups may be the same or different.

As used herein, a C₂ to C₆ alkenyl group is a linear or branched alkenylgroup containing from 2 to 6 carbon atoms and having one or more, e.g.one or two, double bonds. Typically a C₂ to C₆ alkenyl group is a C₂ toC₄ alkenyl group, e.g. a C₂ to C₃ alkenyl group. Examples of C₂ to C₆alkenyl groups include ethenyl, propenyl, butenyl, pentenyl and hexenyl.For the avoidance of doubt, where two alkenyl groups are present, thealkenyl groups may be the same or different.

By way of example, in the methods of the invention, the linear polyamincbackbone is often polylysine with degree of polymerization of 22-450,more often 35-150, still more often 35-85 based on light scattering ornuclear magnetic resonance analysis; the protective chain is oftenmethoxy PEG chain with single carboxyl terminus having 2-20 kDa, moreoften 4-12 kDa, still more often 4-6 kDa number average molecular weightor Mn based on gel permeation chromatography; and the long chain fattyacid-NHS is often C18-NHS.

The following Examples illustrate the invention. They do not however,limit the invention in any way. In this regard, it is important tounderstand that the particular assays used in the Examples section aredesigned only to provide an indication of binding capacity of thepeptide. There are many assays available to determine such capacity, anda negative result in any one particular assay is therefore notdeterminative.

EXAMPLES

The Examples herein describe processes of synthesis that give asemi-random graft co-polymer product that is an efficient binder of amodel peptide called Atrial Natriuretic Peptide (ANP; sequence based onconvention known in the art: SEQ ID NO: 1 SLRRSSCFGGRMDRIGAQSGLGCNSFRYwith intrapeptide disulfide bond). The term “efficient binder” isdefined as a binding characteristic such that when peptide is mixed withthe semi-random graft co-polymer product at a weight ratio of 1:10(Peptide:Co-polymer, wt:wt) in a physiological or near physiologicalbuffer the amount of free peptide (evaluated as described below) will beless than 12%. Such a binder does not exist prior to the disclosure ofthe present disclosure.

It should be noted that, for any given co-polymer binder, as the weightratio of peptide to co-polymer increases the amount of free peptideincreases and, conversely, as the weight ratio of peptide to co-polymerdecreases the amount of free peptide decreases. This is because theco-polymer, the subject of the present disclosure, is a reversiblebinder that has properties that include capacity. If the capacity issaturated by peptide due to higher loading, any additional peptide willnot bind. Because of the complexity of the structure of the groups ofco-polymer, the subject of the present disclosure, the only way todistinguish differences in composition is by comparing their measurableproperties.

General Reagents

Unless otherwise indicated, reagents were used without furtherpurification. In addition, the reagents that were used are commerciallyavailable and their syntheses are well known in the art.

Poly-L-lysine hydrobromide (PL), DPn by NMR 55, Mm by NMR 11,500, PDI byGPC 1.04, was from Alamanda Polymers (Huntsville, Ala.);methoxypolyethyleneglycol-carboxyl (MPEG-CM), MW 5 kDa was from LaysanBio Inc (Arab, Ala.); N-hydroxysuccinimidesulfate (NHSS), MW 217 Da wasfrom ChemPep (Wellington, Fla.); methoxypolyethyleneglycol succinimidylsuccinate (MPEG-SCM), MW 5 kDa was from JenKem Technology USA (Plano,Tex.); and 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), MW 192Da was from Pierce (Rockford, Ill.). Stearic acid (C18), MW 284 Da wasfrom Alfa Aesar (Ward Hill Mass.); N-Hydroxysuccinimide (NHS), MW 115 Dawas from Acros Organics (Pittsburgh, Pa.); andN,N′-dicyclohexylcarbodiimide (DCC), MW 206 Da was from Acros Organics(Pittsburgh, Pa.).

General Techniques

The method modified from Lapidot et al. [Lapidot, Y., Rappoport, S. andWolman, Y. (1967) J. Lipid Res., 8, 142] was used to prepare stearicacid N-hydroxysuccinimide ester (C18-NHS), MW 382 Da. Stearic acid (MW284 Da, 17 gm, 60 mmol) in 230 mL ethyl acetate was activated with NHS(MW 115 Da, 6.9 g, 60 mmol) and DCC (MW 206 Da, 9.6 mL, 12.4 g, 60 mmol)and incubated overnight. The urea precipitate was removed by filtration.The filtrate was then dried and recrystallized using ethanol. Theproduct C18-NHS was collected by filtration, dried under vacuum, andstored frozen desiccated.

Buffers were prepared in deionized distilled water. The adjustment ofreaction pH was performed using a pH meter.

The degree of PL modification by PEG and C18 was measured by assessingthe consumption of free primary amino groups using a TNBS assay (Sparadoet al. (1979) Anal. Biochem. 96, 317). Visible light absorbance ofsamples was measured using a microplate reader.

The product was washed by ultrafiltration through 100 kDa or 50 kDacut-off membrane. Ultrafiltration was performed using membranecartridges (UFP-100-E or UFP-50-E, GE-Amersham Biosciences Corp,Westborough, Mass.) mounted on a QuixStand system (GE-AmershamBiosciences Corp, Westborough, Mass.). Typical washing conditions use 10volume-equivalents (relative to solution volume being washed) of 90%alcohol, 10-15 volumes of 80% alcohol and 10 volumes of water. Theproduct was then filter-sterilized and lyophilized.

The hydrodynamic diameter of graft co-polymers was assessed by Gelpermeation chromatography using TosohG4000WXL HPLC column calibratedusing globular protein standards. Binding to peptide of the graftco-polymer products of various processes was evaluated by 2-hourincubation of the co-polymer with the corresponding peptide in phosphatebuffered saline at pH 7.35 or 100 mM HEPES buffer pH 7.35 followed byfiltration through a 100 kDa molecular weight cut-off membrane filter(regenerated cellulose filter, YM-100, from Millipore, Bedford, Mass.)by centrifugation. No difference in a peptide binding to a graftco-polymer product was observed between these two buffers.

The filtrate containing free (unbound) peptide was quantified by reversephase HPLC (Synergi 2.5 um Max, 0.4×2 cm) monitored at 220 nm ran at aflow rate of 1.5 mL/min using a gradient of 0% B for 1 min and 25-50% Bfor 1-5 min, where A is 5% acetonitrile with 0.1% TFA and B is 100%acetonitrile with 0.1% TFA. The filtrate from control tubes containingpeptide without the co-polymer was taken as the total amount of peptideavailable for binding. The amount of bound peptide can be calculated bysubtracting the concentration of free peptide that passed through thefilter from the corresponding control that received the sameconcentration of peptide but without the graft co-polymer. Binding wastested at various ratios such that 2, 5, and 10% loading represent 1:50,1:20, and 1:10 peptide weight to co-polymer weight ratio.

Methods

Unless otherwise indicated, the synthesis of graft co-polymers wasperformed by preparing a series of solutions (Sol A, Sol B, Sol C, Sol Dand Sol E) and combining them or by adding reagents to them during thePEGylation and Fatylation steps of the chemical process. Sol A containedPL dissolved in buffer (250 or 50 mM HEPES; or 100 mM TEOA) and Sol Bcontained mPEG-CM activated with NHSS and EDC in MES buffer (10 mM, pH4.7) at room temperature. Sol C is the reaction solution wherePEGylation of PL occurs and was prepared at room temperature bycombining Sol A and Sol B, or by adding reagents (NHSS, EDC or PEG-SCM)directly to Sol A, thus having zero minute pre-activation and no Sol B.No solution B is different from having solution B with 0 minpre-activation. When primary amino groups, as measured by TNBS, werefound to be 41-62% Sol D was prepared by adding organic solvent(acetonitrile, acetone, or dichloromethane) to solution C and heating to55-60° C. Sol E is the reaction solution where Fatylation occurs and wasprepared adding C18-NHS, and DIPEA to Sol D. Sol E was allowed to coolto room temperature and allowed to stir from two hours to overnight. Avolume of water approximately equal to the volume of Sol E was added toSol E and the solution extracted 2-3 times with 2 volumes of a polarorganic solvent (e.g., ethyl acetate). The aqueous phase containingproduct was then diluted with distilled deionized water and washed byultrafiltration described above. In some cases the chemical process doesnot require Sol B as in the case where PEG-SCM, EDC or NHSS are added toSol A to produce Sol C. In all cases, equivalents of reagents arereported relative to the starting primary amino content of PL.

Example 1 Samples 1-A, 1-B, and 1-C

Synthesis of graft co-polymer 1-A, 1-B, and 1-C (5 kDa MPEG-CM; 20 kDaPL; 59, 53, and 54% saturation of amino groups with PEG and remainingprimary amino groups modified with stearic acid) using MPEG-CM for thePEGylation reaction. The syntheses were performed as described in theGeneral section unless specified otherwise. Activation of Sol B wasallowed to proceed for 20 minutes. After 2 and 3 hrs. additional EDC wasadded to Sol C (total EDC reported in ratio tables for individual lots).The following tables below provide the properties of the products andthe ratios of each of the reagents used during the synthesis.

TABLE 13 Properties of Synthesized Materials Residual % Free % PEGHydrodynamic Amino ANP Evaluated Sample Saturation Diameter (nm)(nmol/mg) at 10% Loading 1-A 59 18.8 1.2 35* 1-B 53 19.9 1.0 32* 1-C 5420.5 1.1 29* *The corresponding process failed to provide products withthe desired property.

The moderate binding of the synthesized graft co-polymers was not withinthe range of acceptable quantities for free ANP of 13% or less at 10%peptide loading. Thus, the ratio of reagents used for this processprovided product that was inferior compared to the product produced bythe process in the present disclosure.

TABLE 14 Ratios for Sample 1-A in Example 1 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.620 mmol Y = 4 mL bCarboxyl in solution B (moles) 0.771W Z = 8 mL b NHSS in solution B(moles) 0.871W Z = 8 mL b EDC in solution B (moles)  1.58W Z = 8 mL b pHof solution B 4.0-5.5 b Activation time (min) 20 c & d pH of solution C7.7 c & d Amino in solution C (Molar) W/(Y + Z) = 51.7 mM e AdditionalEDC added to 1.87 × W [3.45 × W] solution C [total EDC in C] (moles) fAcetonitrile in Sol D 20.5 mL g DIPEA in solution D (moles) 2.47 × W hC18-NHS in solution E (moles) 1.12 × W Sol D volume + C18- NHS volume =39.7 mL

TABLE 15 Ratios for Sample 1-B in Example 1 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.578 mmol Y = 4 mL bCarboxyl in solution B (moles) 0.827 × W  Z = 8 mL b NHSS in solution B(moles) 0.934 × W  Z = 8 mL b EDC in solution B (moles) 1.41 × W Z = 8mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C 7.7 c & d Amino in solution C (Molar) W/(Y + Z) = 48.2 mM eAdditional EDC added to 2.42 × W [3.83 × W] solution C [total EDC in C](moles) f Acetonitrile in Sol D 20.5 mL g DIPEA in solution D (moles)2.65 × W h C18-NHS in solution E (moles) At least 1.21 × W Sol Dvolume + C18- NHS volume = 39.7 mL

TABLE 16 Ratios for Sample 1-C in Example 1 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.571 mmol Y = 4 mL bCarboxyl in solution B (moles) 0.837 × W  Z = 8 mL b NHSS in solution B(moles) 0.946 × W  Z = 8 mL b EDC in solution B (moles) 1.34 × W Z = 8mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C 7.7 c & d Amino in solution C (Molar) W/(Y + Z) = 47.5 mM eAdditional EDC added to 2.39 × W [3.73 × W] solution C [total EDC in C](moles) f Acetonitrile in Sol D 20.5 mL g DIPEA in solution D (moles)2.68 × W h C18-NHS in solution E (moles) 1.22 × W Sol D volume + C18-NHS volume = 39.7 mL

Example 2 Samples 2-A, 2-B, and 2-C

Synthesis of graft co-polymer 2-A, 2-B, and 2-C (5 KDa MPEG-CM; 20 kDaPL; 45, 44, and 44% saturation of primary amino groups with PEG andremaining primary amino groups modified with stearic acid) using MPEG-CMfor the PEGylation reaction. The syntheses were performed as describedin the General section unless specified otherwise. Activation of Sol Bwas allowed to proceed for 20 minutes. After 2 and 3 hrs. additional EDCwas added to Sol C (total EDC reported in ratio tables for individuallots).

TABLE 17 Properties of Synthesized Materials Residual % Free % PEGHydrodynamic Amino ANP Evaluated Sample Saturation Diameter (nm)(nmol/mg) at 10% Loading 2-A 45 19.3 0.8 34* 2-B 44 19.3 0.9 32* 2-C 4419.9 1.4 37* *The corresponding process failed to provide products withthe desired property.

The moderate binding of the synthesized graft co-polymers was not withinthe range of acceptable quantities for free ANP of 13% or less at 10%peptide loading. Thus, the ratio of reagents used for this processprovided product that was inferior compared to the product produced bythe process in the present disclosure.

TABLE 18 Ratios for Sample 2-A in Example 2 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.536 mmol Y = 4 mL bCarboxyl in solution B (moles) 0.803 × W  Z = 7.3 mL b NHSS in solutionB (moles) 0.926 × W  Z = 7.3 mL b EDC in solution B (moles) 1.61 × W Z =7.3 mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C 7.7 c & d Amino in solution C (Molar) W/(Y + Z) = 47.4 mM eAdditional EDC added to solution 1.94 × W [3.55 × W] C [total EDC in C](moles) f Acetonitrile in Sol D 20 mL g DIPEA in solution D (moles) 2.62× W h C18-NHS in solution E (moles) 1.19 × W Sol D volume + C18- NHSvolume = 37.4 mL

TABLE 19 Ratios for Sample 2-B in Example 2 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.539 mmol Y = 4 mL bCarboxyl in solution B (moles) 0.799 × W  Z = 7.3 mL b NHSS in solutionB (moles) 0.921 × W  Z = 7.3 mL b EDC in solution B (moles) 1.60 × W Z =7.3 mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C 7.7 c & d Amino in solution C (Molar) W/(Y + Z) = 47.7 mM eAdditional EDC added to solution 1.94 [3.54 × W] C [total EDC in C](moles) f Acetonitrile in Sol D 20 mL g DIPEA in solution D (moles) 2.61× W h C18-NHS in solution E (moles) At least 1.18 × W Sol D volume +C18- NHS volume = 37.4 mL

TABLE 20 Ratios for Sample 2-C in Example 2 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.548 mmol Y = 4 mL bCarboxyl in solution B (moles) 0.759 × W  Z = 7.1 mL b NHSS in solutionB (moles) 0.875 × W  Z = 7.1 mL b EDC in solution B (moles) 1.52 × W Z =7.1 mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C 7.7 c & d Amino in solution C (Molar) W/(Y + Z) = 49.3 mM eAdditional EDC added to 1.92 × W [3.44 × W] solution C [total EDC in C](moles) f Acetonitrile in Sol D 20 mL g DIPEA in solution D (moles) 2.56× W h C18-NHS in solution E (moles) 1.16 × W Sol D volume + C18- NHSvolume = 37.2 mL

Example 3 Samples 3-A, 3-B, and 3-C

Synthesis of graft co-polymer 3-A, 3-B, and 3-C (5 kDa MPEG-CM; 20 kDaPL; 54, 60, and 56% saturation of primary amino groups with PEG andremaining primary amino groups modified with stearic acid) using MPEG-CMfor the PEGylation reaction. The syntheses were performed as describedin the General section unless specified otherwise. Activation of Sol Bwas allowed to proceed for 20 minutes. After 2 and 3 hrs additional EDCwas added to Sol C (total EDC reported in ratio tables for individuallots).

TABLE 21 Properties of Synthesized Materials Residual % Free % PEGHydrodynamic Amino ANP Evaluated at Sample Saturation Diameter (nm)(nmol/mg) 10% Loading 3-A 54 18.0 1.1 24* 3-B 60 17.7 1.2 25* 3-C 5618.8 1.3 22* *The corresponding process failed to provide products withthe desired property.

The moderate binding of the synthesized graft co-polymers was not withinthe range of acceptable quantities for free ANP of 13% or less at 10%peptide loading. Thus, the ratio of reagents used for this processprovided product that was inferior compared to the product produced bythe process in the present disclosure.

TABLE 22 Ratios for Sample 3-A in Example 3 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.536 mmol Y = 4 mL bCarboxyl in solution B (moles) 0.890 × W  Z = 7.9 mL b NHSS in solutionB (moles) 1.03 × W Z = 7.9 mL b EDC in solution B (moles) 1.79 × W Z =7.9 mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C 7.7 c & d Amino in solution C (Molar) W/(Y + Z) = 45.0 mM eAdditional EDC added to solution C 2.13 × W [3.92 × W] [total EDC in C](moles) f Acetonitrile in Sol D 20 mL g DIPEA in solution D (moles) 2.33× W h C18-NHS in solution E (moles) 1.08 × W Sol D volume + C18- NHSvolume = 37.8 mL

TABLE 23 Ratios for Sample 3-B in Example 3 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.539 mmol Y = 4 mL bCarboxyl in solution B (moles) 0.885 × W  Z = 7.9 mL b NHSS in solutionB (moles) 1.02 × W Z = 7.9 mL b EDC in solution B (moles) 1.78 × W Z =7.9 mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C 7.7 c & d Amino in solution C (Molar) W/(Y + Z) = 45.3 mM eAdditional EDC added to 2.15 × W [3.92 × W] solution C [total EDC in C](moles) f Acetonitrile in Sol D 20 mL g DIPEA in solution D (moles) 2.32× W h C18-NHS in solution E (moles) 1.07 × W Sol D volume + C18-NHSvolume = 37.8 mL

TABLE 24 Ratios for Sample 3-C in Example 3 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.548 mmol Y = 4 mL bCarboxyl in solution B (moles) 0.870 × W  Z = 7.9 mL b NHSS in solutionB (moles) 1.01 × W Z = 7.9 mL b EDC in solution B (moles) 1.75 × W Z =7.9 mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C 7.7 c & d Amino in solution C (Molar) W/(Y + Z) = 46.1 mM eAdditional EDC added to solution 2.11 × W [3.86 × W] C [total EDC in C](moles) f Acetonitrile in Sol D 20 mL g DIPEA in solution D (moles) 2.38× W h C18-NHS in solution E (moles) 1.06 × W Sol D volume + C18-NHSvolume = 37.8

Example 4 Samples 4-A, 4-B, and 4-C

Synthesis of graft co-polymer 4-A, 4-B, and 4-C (5 kDa MPEG-CM; 20 kDaPL; 53, 51, and 47% saturation of primary amino groups with PEG andremaining primary amino groups modified with stearic acid) using MPEG-CMfor the PEGylation reaction without preactivation. The syntheses wereperformed as described in the General section unless specifiedotherwise. Sol B was not prepared; reagents were added directly to SolA.

TABLE 25 Properties of Synthesized Materials. Residual % Free % PEGHydrodynamic Amino ANP Evaluated Sample Saturation Diameter (nm)(nmol/mg) at 10% Loading 4-A 53 22.9 0 21* 4-B 51 22.3 0 19* 4-C 47 22.90 16* *The corresponding process failed to provide products with thedesired property.

The moderate binding of the synthesized graft co-polymers was not withinthe range of acceptable quantities for free ANP of 13% or less at 10%peptide loading. Thus, the ratio of reagents and method of addition usedfor this process provided product that was inferior compared to theproduct produced by the process in the present disclosure.

TABLE 26 Ratios for Sample 4-A in Example 4 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.537 mmol Y = 4 mL bCarboxyl in solution C (moles) 0.839 × W  Z = 8 mL b NHSS in solution C(moles) 0.995 × W  Z = 8 mL b EDC in solution C (moles) 2.50 × W Z = 8mL b pH of solution B not applicable b Activation time (min) 0 c & d pHof solution C 7.7 c & d Amino in solution C (Molar) W/(Y + Z) = 44.8 mMe Additional EDC added to solution No additional EDC C [total EDC in C](moles) added f Acetonitrile in Sol D 21 mL g DIPEA in solution D(moles) 2.52 × W h C18-NHS in solution E (moles) 1.18 × W Sol D volume +C18- NHS volume = 39.4 mL

TABLE 27 Ratios for Sample 4-B in Example 4 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.517 mmol Y = 4 mL bCarboxyl in solution B (moles) 0.870 × W  Z = 8 mL b NHSS in solution B(moles) 1.06 × W Z = 8 mL b EDC in solution B (moles) 2.59 × W Z = 8 mLb pH of solution B not applicable b Activation time (min) 0 c & d pH ofsolution C 7.7 c & d Amino in solution C (Molar) W/(Y + Z) = 43.1 mM eAdditional EDC added to solution No additional EDC C [total EDC in C](moles) added f Acetonitrile in Sol D 21 mL g DIPEA in solution D(moles) 2.62 × W h C18-NHS in solution E (moles) 1.23 × W Sol D volume +C18- NHS volume = 39.4 mL

TABLE 28 Ratios for Sample 4-C in Example 4 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.492 mmol Y = 4 mL bCarboxyl in solution C (moles) 0.914 × W  Z = 8 mL b NHSS in solution C(moles) 1.09 × W Z = 8 mL b EDC in solution C (moles) 2.66 × W Z = 8 mLb pH of solution B not applicable b Activation time (min) 0 c & d pH ofsolution C 7.7 c & d Amino in solution C (Molar) W/(Y + Z) = 41.0 mM eAdditional EDC added to No additional EDC solution C [total EDC in C]added (moles) f Acetonitrile in Sol D 21 mL g DIPEA in solution D(moles) 2.75 × W h C18-NHS in solution E (moles) 1.29 × W Sol D volume +C18- NHS volume = 39.4 mL

Example 5 Samples 5-A, 5-B, and 5-C

Synthesis of graft co-polymer 5-A, 5-B, and 5-C (5 KDa MPEG-CM; 20 kDaPL; 57, 61, and 55% saturation of primary amino groups with PEG andremaining primary amino groups modified with stearic acid) using MPEG-CMfor the PEGylation reaction without preactivation. The syntheses wereperformed as described in the General section unless specifiedotherwise. Sol B was not prepared; reagents were added directly to SolA.

TABLE 29 Properties of Synthesized Materials Residual % Free % PEGHydrodynamic Amino ANP Evaluated Sample Saturation Diameter (nm)(nmol/mg) at 10% Loading 5-A 57 22.9 0 18* 5-B 61 22.3 0.1 17* 5-C 5522.9 0.2 15* *The corresponding process failed to provide products withthe desired property.

The moderate binding of the synthesized graft co-polymers were notwithin the range of acceptable quantities for free ANP of 13% or less at10% peptide loading. Thus, the ratio of reagents and the method ofaddition used for this process provided product that was inferiorcompared to the product produced by the process in the presentdisclosure.

TABLE 30 Ratios for Sample 5-A in Example 5 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.542 mmol Y = 4 mL bCarboxyl in solution C (moles) 0.821 × W  Z = 8 mL b NHSS in solution C(moles) 0.948 × W  Z = 8 mL b EDC in solution C (moles) 2.41 × W Z = 8mL b pH of solution B not applicable b Activation time (min) 0 c & d pHof solution C 7.7 c & d Amino in solution C (Molar) W/(Y + Z) = 45.2 mMe Additional EDC added to solution No additional EDC C [total EDC in C](moles) added f Acetonitrile in Sol D 21.5 mL g DIPEA in solution D(moles) 2.50 × W h C18-NHS in solution E (moles) 1.16 × W Sol D volume +C18- NHS volume = 39.9 mL

TABLE 31 Ratios for Sample 5-B in Example 5 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.601 mmol Y = 4 mL bCarboxyl in solution C (moles) 0.741 × W  Z = 8 mL b NHSS in solution C(moles) 0.849 × W  Z = 8 mL b EDC in solution C (moles) 2.20 × W Z = 8mL b pH of solution B not applicable b Activation time (min) 0 c & d pHof solution C 7.7 c & d Amino in solution C (Molar) W/(Y + Z) = 50.1 mMe Overall EDC added to solution C No additional EDC after 2 hours(moles), 2 more added additions at 1 h intervals f Acetonitrile in Sol D21.5 mL g DIPEA in solution D (moles) 2.25 × W h C18-NHS in solution E(moles) 1.04 × W Sol D volume + C18- NHS volume = 39.9 mL

TABLE 32 Ratios for Sample 5-C in Example 5 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.604 mmol Y = 4 mL bCarboxyl in solution C (moles) 0.737 × W  Z = 8 mL b NHSS in solution C(moles) 0.864 × W  Z = 8 mL b EDC in solution C (moles) 2.19 × W Z = 8mL b pH of solution B not applicable b Activation time (min) 0 c & d pHof solution C 7.7 c & d Amino in solution C (Molar) W/(Y + Z) = 50.3 mMe Additional EDC added to solution No additional EDC C [total EDC in C](moles) added f Acetonitrile in Sol D 21.5 mL g DIPEA in solution D(moles) 2.24 × W h C18-NHS in solution E (moles) 1.04 × W Sol D volume +C18- NHS volume = 39.9 mL

Example 6 Samples 6-A, 6-B, and 6-C

Synthesis of graft co-polymer 6-A, 6-B, and 6-C (5 kDa MPEG-CM; 20 kDaPL; 99, 98, and 97% saturation of primary amino groups with PEG andremaining primary amino groups modified with stearic acid) using MPEG-CMfor the PEGylation reaction without preactivation. The synthesis wasperformed as described in the General section unless specifiedotherwise. Sol B was not prepared; reagents were added directly to SolA.

TABLE 33 Properties of Synthesized Materials Residual % Free % PEGHydrodynamic Amino ANP Evaluated at Sample Saturation Diameter (nm)(nmol/mg) 10% Loading 6-A 99 15.2 2.9 22* 6-B 98 14.7 3.7 27* 6-C 9716.7 5.4 14* *The corresponding process failed to provide products withthe desired property.

The moderate binding of the synthesized graft co-polymers was not withinthe range of acceptable quantities for free ANP of 13% or less at 10%peptide loading. The ratio of reagents used for the samples ofexperiment 6 was similar to the ratio of reagents used for graftco-polymer lots that produced acceptable binding characteristics (see,samples of experiments 7, 8, and 9), however only 6C was close tomeeting ANP binding criteria. Additionally, the % PEG saturation wasdifficult to control due to precipitation and reached well above the 62%range described in the General procedure; the apparent saturation may bean artifact of precipitation as amino groups of PL would not beavailable for detection by TNBS if PL precipitated. Presumablypreparation of Sol B (preactivation of PEG-SCM with NHSS and EDC in MESbuffer at pH 4.0 to 5.5) is necessary for reproducibly preparing graftco-polymers with suitable binding characteristic. Thus, the processprovided product that was inferior compared to the product produced bythe process in the present disclosure although it is believed thatacceptable product can be produced if the conditions leading toprecipitation were avoided by adding powder reagents to solution A undervigorous stirring condition, a process that is different from failedExample 6 (Samples 6-A, 6-B, and 6-C).

TABLE 34 Ratios for Sample 6-A in Example 6 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.566 mmol Y = 4 mL bCarboxyl in solution C (moles) 0.875 × W  Y + Z = 12 mL b NHSS insolution C (moles) 2.64 × W Y + Z = 12 mL b EDC in solution C (moles)3.51 × W Y + Z = 12 mL b pH of solution C 7.7 b Activation time (min) 0c & d Amino in solution C (Molar) W/(Y + Z) = 47.2 mM e Additional EDCadded to solution No additional EDC C [total EDC in C] (moles) added fAcetonitrile in Sol D 22 mL g DIPEA in solution D (moles) 2.35 × W hC18-NHS in solution E (moles) 1.08 × W Sol D volume + C18- NHS volume =40.6 mL

TABLE 35 Ratios for Sample 6-B in Example 6 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.530 mmol Y = 4 mL bCarboxyl in solution C (moles) 0.934 × W  Y + Z = 12 mL b NHSS insolution C (moles) 2.80 × W Y + Z = 12 mL b EDC in solution C (moles)3.74 × W Y + Z = 12 mL b pH of solution C 7.7 b Activation time (min) 0c & d Amino in solution C (Molar) W/(Y + Z) = 44.2 mM e Additional EDCadded to solution No additional EDC C [total EDC in C] (moles) added fAcetonitrile in Sol D 22 mL g DIPEA in solution D (moles) 2.51 × W hC18-NHS in solution E (moles) 1.15 × W Sol D volume + C18- NHS volume =40.6 mL

TABLE 36 Ratios for Sample 6-C in Example 6 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.656 mmol Y = 4 mL bCarboxyl in solution C (moles) 0.754 × W  Y + Z = 12 mL b NHSS insolution C (moles) 2.27 × W Y + Z = 12 mL b EDC in solution C (moles)3.01 × W Y + Z = 12 mL b Activation time (min) 0 c & d pH of solution C7.7 c & d Amino in solution C (Molar) W/(Y + Z) = 54.7 mM e AdditionalEDC added to solution No additional EDC C [total EDC in C] (moles) addedf Acetonitrile in Sol D 22 mL g DIPEA in solution D (moles) 2.03 × W hC18-NHS in solution E (moles) 0.93 × W Sol D volume + C18-NHS volume =40.6 mL

Example 7 Samples 7-A, 7-B, and 7-C

Synthesis of graft co-polymer 7-A, 7-B, and 7-C (5 kDa MPEG-CM; 20 kDaPL; 58, 55, and 55% saturation of primary amino groups with PEG andremaining primary amino groups modified with stearic acid) using MPEG-CMfor the PEGylation reaction. The syntheses were performed as describedin the General section unless specified otherwise. Activation of Sol Bwas allowed to proceed for 20 minutes. After 2 hours additional EDC wasadded to Sol C (total EDC reported in ratio tables for individual lots).

TABLE 37 Properties of Synthesized Materials Residual % Free % PEGHydrodynamic Amino ANP Evaluated at Sample Saturation Diameter (nm)(nmol/mg) 10% Loading 7-A 58 22.3 5.6 11* 7-B 55 23.0 13.9 12* 7-C 5523.0 7.2  7* *The corresponding processes produce products that areefficient binders which is the desired property.

The synthesized graft co-polymers produced binding results of acceptablequantities for free ANP of 13% or lower at 10% peptide loading. Thus,the ratio of reagents used for the process in the present disclosureprovided product that was acceptable.

TABLE 38 Ratios for Sample 7-A in Example 7 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.545 mmol Y = 4 mL bCarboxyl in solution B (moles) 0.930 × W  Z = 8.5 mL b NHSS in solutionB (moles) 2.77 × W Z = 8.5 mL b EDC in solution B (moles) 2.80 × W Z =8.5 mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C 7.8 c & d Amino in solution C (Molar) W/(Y + Z) = 43.6 mM eAdditional EDC added to solution 0.94 × W [3.74 × W] C [total EDC in C](moles) f Acetonitrile in Sol D 22 mL g DIPEA in solution D (moles) 1.84× W h C18-NHS in solution E (moles) 0.896 × W  Sol D volume + C18- NHSvolume = 39.9 mL

TABLE 39 Ratios for Sample 7-B in Example 7 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.553 mmol Y = 4 mL bCarboxyl in solution B (moles) 0.916 × W  Z = 8.5 mL b NHSS in solutionB (moles) 2.73 × W Z = 8.5 mL b EDC in solution B (moles) 2.76 × W Z =8.5 mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C 7.8 c & d Amino in solution C (Molar) W/(Y + Z) = 44.1 mM eAdditional EDC added to solution 0.97 [3.73 × W] C [total EDC in C](moles) f Acetonitrile in Sol D 22 mL g DIPEA in solution D (moles) 2.07× W h C18-NHS in solution E (moles) 0.949 × W  Sol D volume + C18- NHSvolume = 39.9 mL

TABLE 40 Ratios for Sample 7-C in Example 7 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.544 mmol Y = 4 mL bCarboxyl in solution B (moles) 0.931 × W Z = 8.5 mL b NHSS in solution B(moles) 2.80 × W Z = 8.5 mL b EDC in solution B (moles) 2.51 × W Z = 8.5mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C 7.8 c & d Amino in solution C (Molar) W/(Y + Z) = 43.5 mM eAdditional EDC added to 1.07 × W [3.88 × W] solution C [total EDC in C](moles) f Acetonitrile in Sol D 22 mL g DIPEA in solution D (moles) 2.11× W h C18-NHS in solution E (moles) 0.965 × W  Sol D volume + C18- NHSvolume = 39.9 mL

Example 8 Samples 8-A, 8-B, and 8-C

Synthesis of graft co-polymer 8-A, 8-B, and 8-C (5 KDa MPEG-CM; 20 kDaPL; 59, 51, and 41% saturation of primary amino groups with PEG andremaining primary amino groups modified with stearic acid) using MPEG-CMfor the PEGylation reaction. The syntheses were performed as describedin the General section unless specified otherwise. Activation of Sol Bwas allowed to proceed for 20 minutes. After 2 hours additional EDC wasadded to Sol C (total EDC reported in ratio tables for individual lots).

TABLE 41 Properties of Synthesized Materials Residual % Free % PEGHydrodynamic Amino ANP Evaluated Sample Saturation Diameter (nm)(nmol/mg) at 10% Loading 8-A 59 21.1+ 3.1  8* 8-B 51 23.6 4.5 10* 8-C 4124.2+ 5.9 10* *The corresponding processes produce products that areefficient binder which is the desired property.

The synthesized graft co-polymers produced binding results of acceptablequantities for free ANP of 13% or less at 10% peptide loading. Thus, theratio of reagents used for the process in the present disclosureprovided product that was acceptable.

TABLE 42 Ratios for Sample 8-A in Example 8 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.548 mmol Y = 4 mL bCarboxyl in solution B (moles) 0.886 × W  Z = 8.3 mL b NHSS in solutionB (moles) 2.67 × W Z = 8.3 mL b EDC in solution B (moles) 2.65 × W Z =8.3 mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C 7.8 c & d Amino in solution C (Molar) W/(Y + Z) = 43.6 mM eAdditional EDC added to solution 1.06 × W [3.71 × W] C [total EDC in C](moles) f Acetonitrile in Sol D 22 mL g DIPEA in solution D (moles) 2.44× W h C18-NHS in solution E (moles) 1.12 × W Sol D volume + C18- NHSvolume = 44.6 mL

TABLE 43 Ratios for Sample 8-B in Example 8 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.534 mmol Y = 4 mL bCarboxyl in solution B (moles) 0.910 × W  Z = 8.3 mL b NHSS in solutionB (moles) 2.74 × W Z = 8.3 mL b EDC in solution B (moles) 2.72 × W Z =8.3 mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C 7.8 c & d Amino in solution C (Molar) W/(Y + Z) = 44.1 mM eAdditional EDC added to solution 0.98 × W [3.70 × W] C [total EDC in C](moles) f Acetonitrile in Sol D 22 mL g DIPEA in solution D (moles) 2.51× W h C18-NHS in solution E (moles) 1.15 × W Sol D volume + C18- NHSvolume = 44.6 mL

TABLE 44 Ratios for Sample 8-C in Example 8 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.543 mmol Y = 4 mL bCarboxyl in solution B (moles) 0.894 × W Z = 8.3 mL b NHSS in solution B(moles) 2.69 × W Z = 8.3 mL b EDC in solution B (moles) 2.68 × W Z = 8.3mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C  7.8 c & d Amino in solution C (Molar) W/(Y + Z) = 43.5 mM eAdditional EDC added to solution 1.16 × W [3.84 × W] C [total EDC in C](moles) f Acetonitrile in Sol D 22 mL g DIPEA in solution D (moles) 2.48× W h C18-NHS in solution E (moles) 1.13 × W Sol D volume + C18- NHSvolume = 44.7 mL

Example 9 Samples 9-A, 9-B, 9-C, 9-D, 9-E, and 9-F

Synthesis of graft co-polymer 9-A, 9-B, 9-C, 9-D, 9-E, and 9-F (5 KDaMPEG-CM; 20 kDa PL; 62, 58, 55, 53, 56 and 56% saturation of primaryamino groups with PEG and remaining primary amino groups modified withstearic acid) using MPEG-CM for the PEGylation reaction. The syntheseswere performed as described in the General section unless specifiedotherwise. Activation of Sol B was allowed to proceed for 20 minutes.After 2 hours additional EDC was added to Sol C (total EDC reported inratio tables for individual lots).

TABLE 45 Properties of Synthesized Materials Residual % Free ANP % PEGHydrodynamic Amino Evaluated at Yield Sample Saturation Diameter (nm)(nmol/mg) 10% Loading (g) 9-A 62 23.6 8.5 3* 0.8 9-B 58 23.6 6.6 4* 0.89-C 55 24.2 9.3 6* 0.8 9-D 53 22.3 5.8 1* 5 9-E 56 22.3 8.0 5* 8 9-F 5621.7 5.4 3* 42 *The corresponding processes produce products that areefficient binder which is the desired property

The synthesized graft co-polymers produced binding results of acceptablequantities for free ANP of 13% or less at 10% peptide loading. Thus, theratio of reagents used for the process in the present disclosureprovided product that was acceptable.

TABLE 46 Ratios for Sample 9-A in Example 9 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.559 mmol Y = 4 mL bCarboxyl in solution B (moles) 0.886 × W Z = 8.4 mL b NHSS in solution B(moles) 2.67 × W Z = 8.4 mL b EDC in solution B (moles) 2.66 × W Z = 8.4mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C  7.8 c & d Amino in solution C (Molar) W/(Y + Z) = 45.1 mM eAdditional EDC added to 0.89 × W [3.55 × W] solution C [total EDC in C](moles) f Acetonitrile in Sol D 22 mL g DIPEA in solution D (moles) 2.18× W h C18-NHS in solution E (moles) 1.00 × W Sol D volume + C18- NHSvolume = 40.1 mL

TABLE 47 Ratios for Sample 9-B in Example 9 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.550 mmol Y = 4 mL bCarboxyl in solution B (moles) 0.900 × W Z = 8.4 mL b NHSS in solution B(moles) 2.71 × W Z = 8.4 mL b EDC in solution B (moles) 2.70 × W Z = 8.4mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C  7.8 c & d Amino in solution C (Molar) W/(Y + Z) = 44.4 mM eAdditional EDC added to solution 0.90 × W [3.60 × W] C [total EDC in C](moles) f Acetonitrile in Sol D 22 mL g DIPEA in solution D (moles) 2.22× W h C18-NHS in solution E (moles) 1.02 × W Sol D volume + C18-NHSvolume = 40.1 mL

TABLE 48 Ratios for Sample 9-C in Example 9 Step Amount/value Volume ofsolution a Amino in solution A (moles) W = 0.542 mmol Y = 4 mL bCarboxyl in solution B (moles) 0.914 × W Z = 8.4 mL b NHSS in solution B(moles) 2.74 × W Z = 8.4 mL b EDC in solution B (moles) 2.74 × W Z = 8.4mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C  7.8 c & d Amino in solution C (Molar) W/(Y + Z) = 43.7 mM eAdditional EDC added to solution 0.92 × W [3.60 × W] C [total EDC in C](moles) f Acetonitrile in Sol D 22 mL g DIPEA in solution D (moles) 2.25× W h C18-NHS in solution E (moles) 1.03 × W Sol D volume + C18-NHSvolume = 40.1 mL

TABLE 49 Ratios for Sample 9-D in Example 9 (scale for 8 gram yield)Step Amount/value Volume of solution a Amino in solution A (moles) W =4.6 mmol Y = 35 mL b Carboxyl in solution B (moles) 0.90 × W Z = 70 mL bNHSS in solution B (moles) 2.6 × W Z = 70 mL b EDC in solution B (moles)2.7 × W Z = 70 mL b pH of solution B 4.0-5.5 b Activation time (min) 20c & d pH of solution C  7.8 c & d Amino in solution C (Molar) W/(Y + Z)= 44 mM e Additional EDC added to solution 0.92 × W [3.6W] C [total EDCin C] (moles) f Acetonitrile in Sol D 186 mL g DIPEA in solution D(moles) 2.1 × W h C18-NHS in solution E (moles) 0.99 × W Sol D volume +C18-NHS volume = 337 mL

TABLE 50 Ratios for Sample 9-E in Example 9 (scale for 8 gram yield)Step Amount/value Volume of solution a Amino in solution A (moles) W =4.5 mmol Y = 35 mL b Carboxyl in solution B (moles) 0.90 × W Z = 70 mL bNHSS in solution B (moles) 2.7 × W Z = 70 mL b EDC in solution B (moles)2.8 × W Z = 70 mL b pH of solution B 4.0-5.5 b Activation time (min) 20c & d pH of solution C  7.8 c & d Amino in solution C (Molar) W/(Y + Z)= 43 mM e Additional EDC added to 0.90 × W [3.7W] solution C [total EDCin C] (moles) f Acetonitrile in Sol D 188 mL g DIPEA in solution D(moles) 2.1 × W h C18-NHS in solution E (moles) 0.99 × W Sol D volume +C18- NHS volume = 340 mL

TABLE 51 Ratios for Sample 9-F in Example 9 (scale for 42 gram yield)Step Amount/value Volume of solution a Amino in solution A (moles) W =24 mmol Y = 175 mL b Carboxyl in solution B (moles) 0.83 × W Z = 335 mLb NHSS in solution B (moles) 2.5 × W Z = 335 mL b EDC in solution B(moles) 2.7 × W Z = 335 mL b pH of solution B 4.0-5.5 b Activation time(min) 20 c & d pH of solution C  7.8 c & d Amino in solution C (Molar)W/(Y + Z) = 47 mM e Additional EDC added to solution 0.70 × W [3.4W] C[total EDC in C] (moles) f Acetonitrile in Sol D 900 mL g DIPEA insolution D (moles) 2.2 × W h C18-NHS in solution E (moles) 0.97 × W SolD volume + C18-NHS volume = 1650 mL

Example 10 Samples 10-A, 10-B, and 10-C

Synthesis of graft co-polymer 10-A, 10-B, and 10-C (5 kDa MPEG-CM; 20kDa PL; 49, 56 and 56% saturation of primary amino groups with PEG andremaining primary amino groups modified with stearic acid) using MPEG-CMfor the PEGylation and extraction after PEGylation. The syntheses wereperformed as described in the General section unless specifiedotherwise. Activation of Sol B was allowed to proceed for 20 minutes.After 2, 3, and 4 hrs., additional EDC was added to Sol C (total EDCreported in ratio tables for individual lots). After the PEGylationreaction Sol C was extracted with a polar organic solvent (e.g., ethylacetate) and Sol D was prepared from the aqueous phase of theextraction.

TABLE 52 Properties of Synthesized Materials % Free ANP % PEGHydrodynamic Residual Amino Evaluated at Sample Saturation Diameter (nm)(nmol/mg) 10% Loading 10-A 49 22 4.8 55* 10-B 56 20 6.3 78* 10-C 56 206.3 72* *The corresponding process failed to provide products with thedesired property.

The poor binding of the synthesized graft co-polymers were not withinthe range of acceptable quantities for free ANP of 13% or less at 10%peptide loading. The poor binding characteristics of the synthesizedgraft co-polymers can be attributed to disrupting the process byextracting Sol C with a polar organic solvent (e.g., ethyl acetate).This hypothesis is supported by comparing to similar processes (see,samples of experiments 1, 2, and 3) with the same ratio of reagentswhich produce graft co-polymers with moderate binding characteristics.Thus, the ratio of reagents used for this process and the disruption ofthe process with extraction of Sol C provided product that was inferiorcompared to the product produced by the process in the presentdisclosure.

TABLE 53 Ratios for Sample 10-A in Example 10 Step Amount/value Volumeof solution a Amino in solution A (moles) W = 0.361 mmol Y = 3 mL bCarboxyl in solution B (moles) 0.809 × W Z = 4.9 mL b NHSS in solution B(moles) 0.920 × W Z = 4.9 mL b EDC in solution B (moles) 1.60 × W Z =4.9 mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C  7.7 c & d Amino in solution C (Molar) W/(Y + Z) = 45.7 mM eAdditional EDC added to solution 2.89 × W [4.49 × W] C [total EDC in C](moles) f Acetone in Sol D 70 mL g DIPEA in solution D (moles) 9.67 × Wh C18-NHS in solution E (moles) 4.38 × W Sol D volume + C18- NHS volume= 80 mL

TABLE 54 Ratios for Sample 10-B in Example 10 Step Amount/value Volumeof solution a Amino in solution A (moles) W = 0.361 mmol Y = 3 mL bCarboxyl in solution B (moles) 0.809 × W Z = 4.9 mL b NHSS in solution B(moles) 0.930 × W Z = 4.9 mL b EDC in solution B (moles) 1.60 × W Z =4.9 mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C  7.7 c & d Amino in solution C (Molar) W/(Y + Z) = 45.7 mM eAdditional EDC added to solution 2.89 × W [4.49 × W] C [total EDC in C](moles) f Acetone in Sol D 70 mL g DIPEA in solution D (moles) 9.67 × Wh C18-NHS in solution E (moles) 4.40 × W Sol D volume + C18- NHS volume= 80 mL

TABLE 55 Ratios for Sample 10-C in Example 10 Step Amount/value Volumeof solution a Amino in solution A (moles) W = 0.357 mmol Y = 3 mL bCarboxyl in solution B (moles) 0.818 × W Z = 4.9 mL b NHSS in solution B(moles) 0.930 × W Z = 4.9 mL b EDC in solution B (moles) 1.62 × W Z =4.9 mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C  7.7 c & d Amino in solution C (Molar) W/(Y + Z) = 45.2 mM eAdditional EDC added to solution 2.98 × W [4.60 × W] C [total EDC in C](moles) f Acetone in Sol D 70 mL g DIPEA in solution D (moles) 9.78 × Wh C18-NHS in solution E (moles) 4.46 × W Sol D volume + C18- NHS volume= 80 mL

Example 11 Sample 11-A

Synthesis of graft co-polymer 11-A (5 kDa MPEG-CM; 20 kDa PL; 57%saturation of primary amino groups with PEG and remaining primary aminogroups modified with stearic acid) using MPEG-CM for the PEGylationreaction and stopping the process before Fatylation. The synthesis wasperformed as described in the General section unless specifiedotherwise. Activation of Sol B was allowed to proceed for 20 minutes.Sol C was placed on ice at 1.75 hrs reaction time. At 2 hrs Sol C wasreturned to room temperature and additional EDC was added to Sol C(total EDC reported in ratio tables for individual lots). Sol Ccontinued to react until a saturation of 57% of primary amino groups wasreached at 3.75 hrs total time. Sol C was frozen and lyophilized. Sol Dwas prepared by dissolving the lyophilized material from Sol C intodichloromethane, and Sol D was not heated.

TABLE 56 Properties of Synthesized Material % Free ANP % PEGHydrodynamic Residual Amino Evaluated at Sample Saturation Diameter (nm)(nmol/mg) 10% Loading 11-A 57 27.4 0 13* *The corresponding processfailed to provide products with the desired property.

The moderate binding of the synthesized graft co-polymers was not withinthe range of acceptable quantities for free ANP of 13% or less at 10%peptide loading. The moderate characteristics of the synthesized graftco-polymers can be attributed to disrupting the process bylyophilization of Sol C. This hypothesis is supported by comparing tosimilar processes (see, samples of experiments 7, 8, and 9) with thesame ratio of reagents which produce graft co-polymers with acceptablebinding characteristics; note that the ratios of C18-NHS and DIPEA arenot similar to the process of the present disclosure but that part ofthe process has a minimum requirement that all of the listed lots met.Thus, stopping the process by lyophilization of Sol C and not heatingSol D provided product that was inferior compared to the productproduced by the process in the present disclosure.

TABLE 57 Ratios for Sample 11-A in Example 11 Step Amount/value Volumeof solution a Amino in solution A (moles) W = 2.66 mmol Y = 23.5 mL bCarboxyl in solution B (moles) 0.886 × W Z = 40 mL b NHSS in solution B(moles) 2.70 × W Z = 40 mL b EDC in solution B (moles) 2.66 × W Z = 8.4mL b pH of solution B 4.0-5.5 b Activation time (min) 20 c & d pH ofsolution C  7.8 c & d Amino in solution C (Molar) W/(Y + Z) = 41.9 mM eAdditional EDC added to solution C 0.91 × W [3.57 × W] [total EDC in C](moles) f Dichloromethane in Sol D 75.5 mL g DIPEA in solution D (moles)4.3 × W h C18-NHS in solution E (moles) 2.64 × W Sol D volume + C18- NHSvolume = 105 mL

Example 12 Sample 12-A, PEG-SCM

Synthesis of graft co-polymer 12-A (5 kDa MPEG-SCM; 20 kDa PL; 53%saturation of amino groups with PEG and remaining amino groups modifiedwith stearic acid) using MPEG-SCM for the PEGylation reaction. Thesynthesis was performed as described in the General section unlessspecified otherwise. The PL was dissolved in 50 mM Triethanolamine, pH7.7. Sol B was not used as PEG-SCM was added directly to Sol A. Sol Cwas frozen after PEGylation. C18-NHS was dissolved in acetone.

TABLE 58 Properties of Synthesized Material % Free ANP % PEGHydrodynamic Residual Amino Evaluated at Sample Saturation Diameter (nm)(nmol/mg) 10% Loading 12-A 53 18.2 0 100* *The corresponding processfailed to provide products with the desired property.

The poor binding of the synthesized graft co-polymer was not within therange of acceptable quantities for free ANP of 13% or lower at 10%peptide loading. The exceptionally poor binding characteristics of thesynthesized graft co-polymer can be attributed to the use of the PEG-SCM(NHS ester of 5 KDa PEG) reagent as opposed to prcactivating PEG-CM withNHSS and EDC. This hypothesis is supported by comparing to differentprocesses (see, sample 1-B, 4-A, and 9-C) that produce graft co-polymerswith similar levels of PEG saturations and result in graft co-polymerswith moderate to acceptable binding characteristics. Potentially, thePEG-SCM acylates PL at a different rate than the PEG-CM activated as anNHSS ester and affects PEG organization on PL which ultimately impactsthe binding of ANP. Thus, the process which used PEG-SCM as a reagentfor PEGylation provided product that was inferior compared to theproduct produced by the process in the present disclosure. No ANPbinding was observed for graft co-polymers synthesized using NHSactivation of MPEG-CM.

Evaluation of PGCs in Binding of GLP-1 and BNP Peptides

PGCs synthesized using the process of the present disclosure were alsoevaluated in their binding to GLP-1 and BNP, see table below. Inaddition to acceptable binding to ANP the PGCs also provided acceptablebinding to both GLP-1 and BNP with 0-6% free peptide at 10% loading and0% free peptide at 5% and 2% loading, respectively. This data supportsthat the process in the present disclosure is different and superior tothe processes described in the previous publications (Castillo et al:showing 5% free at 2% loading for GLP-1). In addition, our data supportsthat the process in the present disclosure is different and superior tothe processes described in the U.S. patent application Ser. No.11/613183 and U.S. patent application Ser. No. 11/971482 patentapplications (showing 33% free at 10% loading for GLP-1).

TABLE 59 Binding Properties of Synthesized Material % of free peptide(standard deviation) ANP GLP-1 BNP Sample 10% 10% 5% 2% 10% 5% 2% 9-D0.84 0 (0) 0 (0) 0 (0) 4.06 (0.26) 0 (0) 0 (0) (0.17) 9-E 5.34 0 (0) 0(0) 0 (0) 5.46 (0.46) 0 (0) 0 (0) (0.20) 9-F 3.29 0 (0) 0 (0) 0 (0) 5.86(1.52) 0 (0) 0 (0) (0.19)

Two Step Process (EDC Quench, PL-PEG Isolation)

The synthesis of graft co-polymers can be accomplished by altering theprocess where the PEGylation reaction is stopped and the resultingPL-PEG product could be purified prior to the Fatylation reaction. Thisprocess would consist of preparing a series of solutions (Sol A, Sol B,Sol C, Sol D and Sol E) and combining them or by adding reagents to themduring the PEGylation and Fatylation steps of the chemical process. SolA would contain PL dissolved in buffer (HEPES or TEOA) and Sol B wouldcontain mPEG-CM activated with NHSS and EDC in MES buffer. Sol C wouldbe the reaction solution where PEGylation of PL occurs, prepared bycombining Sol A and Sol B, or by adding reagents (NHSS, EDC or PEG-SCM)directly to Sol A, thus having zero minute preactivation or no Sol B. Insome cases the chemical process does not require Sol B as in the casewhere PEG-SCM is added to Sol A to produce Sol C. Sol D would beprepared by adding organic solvent (acetonitrile, acetone ordichloromethane) to solution C and heating to 55-60° C. Then a strongnucleophile could be added to Sol D to quench remaining EDC.Acetonitrile in Sol D would be removed by aqueous/organic extraction (asin the general procedure for removal of acetonitrile after Fatylation).The aqueous phase containing PL-PEG product can then be diluted withwater and purified via ultrafiltration. The purified PL-PEG productcould then be used to make solution E by dissolving the product in 66%acetonitrile and heating to 55-60° C. after which C18-NHS, and DIPEAwould be added.

Heating Sol D prior to quenching or stopping the PEGylation reaction isessential to preserve reaction rates similar to the process in thepresent disclosure and to produce product with acceptable bindingcharacteristics. The processes used to synthesize PGC in examples 10 and11 (see, samples 10-A, 10-B, 10-C, and 11-A) illustrate the poor outcomewhen the PEGylation reaction was stopped prior to the addition oforganic solvent and heating; these processes resulted in PGCs that donot have acceptable binding. Thus, for a two-step procedure for PGCsynthesis to be successful, heating Sol D prior to quenching thePEGylation reaction must be part of the process.

All of the patents and publications cited herein are hereby incorporatedby reference in their entireties.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the disclosure described herein. Such equivalents areintended to be encompassed by the following claims. Unless otherwiseindicated, the general mathematical rule of rounding off numbers appliesto all the numbers in the present specification with the exception ofmolecular weight of a non-polydisperse molecule such as for example H2O,NHSS, EDC, etc. Polymers are poly-disperse molecules. Whenever a numberis given, the last digit of the number is understood to be the limit ofcertainty and is a result of rounding off of the range of numbers to thenearest last digit of a given number. For example the “5 kDa polymer”means a range between 4.5 kDa to 5.5 kDa since rounding of 4.51-5.49 kDato the nearest thousand is 5 kDa. Another example is 2.1 mmol is a rangebetween of 2.05 to 2.15 mmol. Another example is 5.0 kDa polymer is arange between 4.95 to 5.05 kDa.

1-26. (canceled)
 27. A method of preparing a semi-random graftco-polymer, wherein the semi-random graft co-polymer is a protectivechain and long fatty acid-grafted polyamine, comprising: step (a):dissolving a linear polyamine backbone containing W moles of freeprimary amino groups in an aqueous solvent to provide solution A havinga volume Y; and B1 or B2, wherein: B1 comprises step (b): activating aprotective chain containing 0.5-1.2×W moles of carboxyl groups with1.7-7.0×W moles of N-hydroxysuccinimidesulfate and 1.5-3.6×W moles of1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride in anaqueous solvent, at pH 4-5.5 in a final volume of Z, such that W/(Y+Z)has a ratio of 0-55 mM, for a period of time of up to 30 minutes toprovide a solution B; step (c): adding solution B to solution A withcontinuous mixing to provide solution C; and step (d): adjusting the pHof solution C to above 6.5; and B2 comprises steps (b-d): adding aprotective chain containing 0.5-1.2×W moles of carboxyl group, 1.7-7.0×Wmoles of N-hydroxysuccinimidesulfate, and 1.5-3.6×W moles of1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to solutionA to provide solution C; adjusting the pH of solution C to above 6.5;and step (e): adding 0.5-1.5×W of additional1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to solutionC 2 to 3 hours after steps (b-d); and waiting until the primary aminogroups are 55-40% of W moles.
 28. A method of preparing a semi-randomgraft co-polymer according to claim 27, comprising the steps of: step(a): dissolving a linear polyamine backbone containing W of free primaryamino groups in aqueous solvent that includes pH 7-8 to obtain solutionA having a volume Y; step (b): activating a protective chain containing0.5-1.2×W carboxyl group by mixing it with 1.7-7.0×W ofN-hydroxysuccinimidesulfate and 1.5-3.6×W of1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) in aqueoussolvent at pH 4-5.5 to obtain solution B with a final volume of Z suchthat W/(Y+Z)=30-55 mM and allowing the activation to proceed for 0-30min to obtain solution B; step (c): adding solution B to solution A withcontinuous mixing resulting in a solution C; step (d): adjusting the pHof solution C to above 6.5; and step (e): adding 0.5-1.5×W of additional1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to solutionC 2 to 3 hours after step (d) and waiting until the remaining primaryamino groups are 55-40% of the original primary amino.
 29. The method ofclaim 27, further comprising the steps of: if absent, step (e): adding0.5-1.5×W moles of additional1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to solutionC 2 to 3 hours after the pH adjustment in step (d); and waiting untilthe primary amino groups are 55-40% of W moles; step (f): adding atleast W moles of a strong nucleophile to solution C and purifying aresulting immediate reaction product by ultrafiltration followed bylyophilization, and dissolving the immediate reaction product inacetonitrile to provide solution D, wherein the strong nucleophilecomprises any one of hydroxyl amine, NaOR, LiR, NaOH, KOH, NaCCR, NaNH₂,NaNHR, NaNR₂, NaI, LiBr, KI, and NaN₃, wherein R is C₁-C₆ alkyl or C₂-C₆alkenyl; step (g): adding 0.5-6×W moles of N,N-diisopropylethylamine orother tertiary amine to solution D; and step (h): adding at least 0.75×Wmoles of a long chain fatty acid-N-hydroxysuccinimide ester to providesolution E and stirring solution E at room temperature for at least 2hours until the primary amino groups are less than 5% W moles to obtaincrude final product.
 30. The method of claim 27, further comprising thesteps of: if absent, step (e): adding 0.5-1.5×W moles of additional1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to solutionC 2 to 3 hours after the pH adjustment in step (d); and waiting untilthe primary amino groups are 55-40% of W moles; step (f) increasingtotal volume of solution C when the primary amino groups are 55-40% of Wmoles by adding 1.0-2.5 volume equivalent of acetonitrile to obtainsolution D and heating solution D to 40-70° C. for at least 10 minutes,adding a strong nucleophile comprising anyone of hydroxyl amine, NaOR,LiR, NaOH, KOH, NaCCR, NaNH₂, NaNHR, NaNR₂, NaI, LiBr, KI, and NaN₃,wherein R is C₁-C₆ alkyl or C₂-C₆ alkenyl; then purifying a resultingintermediate product by ultrafiltration and lyophilization to provide apure intermediate product; and reconstituting the pure immediate productin 66% acetonitrile to provide a solution D; step (g): adding 0.5-6×Wmoles of N,N-diisopropylethylamine or other tertiary amine to_solutionD; and step (h): adding at least 0.75×W moles of long chain fatty acidN-hydroxysuccinimide ester in 40-70° C. acetonitrile to provide solutionE and stirring solution E at room temperature for at least 2 hours oruntil the primary amino groups are less than 5% of W moles to obtain acrude final product.
 31. The method of claim 27, further comprising thesteps of: if absent, step (e): adding 0.5-1.5×W moles of additional1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to solutionC 2 to 3 hours after the pH adjustment in step (d); and waiting untilthe primary amino groups are 55-40% of W moles; step (f): increasingtotal volume of solution C when the remaining primary amino groups is55-40% of the original amino by adding 1.0-2.5 volume equivalent ofacetonitrile to obtain solution D and heating solution D to 40-70° C.,inclusive; step (g): adding 0.5-6×W N,N-diisopropylethylamine (DIPEA) orother tertiary amine to solution D; and step (h): adding at least 0.75×Wequivalent of long chain fatty acid-N-hydroxysuccinimide ester (NHS) in40-70° C. acetonitrile to obtain solution E and stirring solution E atroom temperature for at least 2 hours or until the remaining primaryamino groups are less than 5% of the original primary amino to obtainthe crude final product. wherein the linear polyamine backbone ispolylysine with a degree of polymerization of 35-150, as determined bylight scattering or nuclear magnetic resonance analysis; and wherein theprotective chain is methoxypolyethyleneglycol chain with a singlecarboxyl terminus and has a number average molecular weight of 4-12 kDa,as determined by gel permeation chromatography.
 32. The method of claim27, wherein in B1, step (b) comprises activating the protective chaincontaining 0.5-1.1×W moles of carboxyl groups with 1.7-3.7×W moles ofN-hydroxysuccinimidesulfate and 1.5-3.3×W moles of1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride in anaqueous solvent; and B1 further comprises step (e), adding 0.5-1.2×Wmoles of additional 1-ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride to solution C 2 to 3 hours after the pH adjustment in step(d); and wherein in B2, steps (b-d) comprise adding the protective chaincontaining 0.5-1.1×W moles of carboxyl groups; 1.7-3.7×W moles ofN-hydroxysuccinimidesulfate; and 1.5-3.3×W moles of1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride to solution Ato provide solution C; and step (e) comprises adding 0.5-1.2×W moles ofadditional 1-ethyl-3-dimethylaminopropyl]carbodiimide hydrochloride tosolution C 2 to 3 hours after steps (b-d).
 33. The method of claim 27,wherein in B1, step (b) comprises activating the protective chaincontaining 0.5-1.0×W-moles of carboxyl groups with 1.7-3.4×W moles ofN-hydroxysuccinimidesulfate and 1.5-3.0×W moles of1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride in anaqueous solvent; and step (e) comprises adding 0.5-1.1×W moles ofadditional 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochlorideto solution C 2 to 3 hours after the pH adjustment in step (d); andwherein in B2, steps (b-d) comprise adding the protective chaincontaining 0.5-1.0×W moles of carboxyl groups; 1.7-3.4×W moles ofN-hydroxysuccinimidesulfate; and 1.5-3.0×W moles of1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride to solution Ato provide solution C; and step (e) comprises adding 0.5-1.1×W moles ofadditional 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochlorideto solution C 2 to 3 hours after steps (b-d).
 34. The method accordingto claim 29, further comprising steps: step (i): extracting theacetonitrile and excess fatty acids from the crude final product insolution E using ethyl acetate and discarding the ethyl acetate layerand repeating the extraction of the water layer at least once; and step(j): washing the water layer containing the final product byultrafiltration by at least 10 volume changes of ethanol and water. 35.The method of claim 34, further comprising step (k) freezing the finalproduct.
 36. The method of claim 34, further comprising step (k)lyophilizing the final product.
 37. A method according to claim 29,further comprising steps: (i) exchanging the organic solvent in solutionE containing the final product into water; and (j) washing the finalproduct by ultrafiltration by at least 10 volume changes of ethanol andwater.
 38. A composition comprising a semi-random graft co-polymerobtainable by the method of claim
 27. 39. The composition of claim 38,wherein the semi-random graft co-copolymer is selected based on bindingto glucagon like peptide-1 and/or atrial natriuretic peptide, andderivatives thereof.
 40. A pharmaceutical composition comprising asemi-random graft copolymer of claim 38.